title: Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy.
abstract: Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.
authors.affiliation: Biotechnology Center, University of Technology Dresden, 01307 Dresden, Germany.
authors.forename: David A
authors.initials: DA
authors.lastname: Cisneros
authors.forename: Carlos
authors.initials: C
authors.lastname: Hung
authors.forename: Clemens M
authors.initials: CM
authors.lastname: Franz
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Muller
mesh_terms: D000818:Animals; D002417:Cattle; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D024022:Fibrillar Collagens / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D012867:Skin / Q000378:metabolism; D013997:Time Factors
chemical_list: D024022:Fibrillar Collagens; D003094:Collagen