Temporal Assembly of Collagen Type II Studied by Atomic Force Microscopy

Abstract

Collagen is a key structural protein in several types of tissues including skin, bone and cartilage. [1]. The collagen family currently includes twenty-six different types of collagen, [2] which can be divided into fibril-forming collagens and fibrilassociated collagens with interrupted triple helices (FACITcollagens). [3] Collagen Type II is one of the fibril-forming collagens and the predominant type of collagen in cartilage [1] . The fibril-forming collagens, including collagen Type II, display a characteristic D-band structure due to a staggered arrangement of the molecules in the fibrils. [4] The fibrils are formed by the assembly of tropocollagen molecules, which consist of three polypeptide strands in a triple helical arrangement. In-vitro studies of the nanoscale molecular assembly of collagen Type II is of great interest, since such studies provide fundamental microscopic insights into how the fibrils assemble in vivo. Furthermore, an understanding of the collagen Type II assembly represents an important first initial step on the road to the use of collagenous scaffolds in tissue engineering that might be used for cartilage replacement surgery in the future. Finally, the hierarchical self-assembly of collagen provides a methodology for constructing molecular systems into functional macroscopic materials with well-organized structures.Most knowledge about the fibrillation of fibril-forming collagens have been obtained by studying collagen Type I through a variety of techniques, such as X-ray diffraction, [5] transmission electron microscopy (TEM) [6][7][8][9] and atomic force microscopy (AFM). [10][11][12][13] From these studies it has been concluded that the fibrillation process is entropy driven, [14] and that the formation of D-banded fibrils is observed at temperatures above 21°C. While the formation of collagen Type I is fairly well understood, this is not the case for collagen Type II. [3,[15][16][17] In vivo collagen Type II is secreted as procollagen molecules that are enzymatically processed by the removal of their C-and N-propeptides before or during fibril assembly in the extracellular matrix. [17] The assembly process is influenced by interaction with small proteoglycans and other matrix proteins such as cartilage oligomeric matrix protein (COMP), collagen type IX and collagen type XI. [3,[18][19][20][21] However, other factors such as pH, electrolytes, molecule concentration and temperature are critical environmental parameters for the fibrillation process. [7,22] In the present study we have used high-resolution AFM to study in vitro the hierarchical self-assembly of collagen Type II and to investigate how the temporal collagen assembly process is influenced by the incubation time, temperature, pH and the collagen Type II concentration under physiological buffer conditions. Three different collagen Type II assembly regimes that result in D-banded fibrils through distinct hierarchical supramolecular assemblies are revealed. Experimental SectionSample preparations were performed using commerci...

Graphene Oxide Hybridized nHAC/PLGA Scaffolds Facilitate the Proliferation of MC3T3-E1 Cells

“…For example, collagen type II is one of the fibril-forming collagens and the predominant type of collagen in cartilage. Coordinating collagen type II into the scaffolds may be able to facilitate cartilage bone regeneration [ 61 ]. In addition, the collagen with proper annealing may further strength the scaffolds, which may induce a new composite material with functional structures.…”

Nanoscale measurements of the assembly of collagen to fibrils

“…The process of collagen fibrillation is known to be a strong function of the external pH [17,18,30]. Subsequently we were able to form collagen fibrils by merely increasing the environmental pH.…”

“…The effect of pH and electrolytes on the temporal assembly of collagen was followed by time lapse imaging [16] and the lateral assembly to broad sheet-like fibrillar structures was studied using hydrodynamic flow [17]. AFM was also used to investigate how type II collagen assembly is influenced by incubation, temperature and pH in a physiological buffer [18]. However, studies on the process of fibril formation begins from the level of single molecules have still not been elucidated satisfactorily.…”

In Vitro Analysis of the Co-Assembly of Type-I and Type-III Collagen

“…15 Among 28 known collagen types, 44 the fibrillar ones including types-I, II, III, and V are dominant, and their assembly into fibrils and fibrillar networks have been extensively studied. 1,7,10,42 Collagen is also used for a wide range of biomedical and biotechnological applications, including templates for fabricating nanowires, 46 as a coating material for non-biological surfaces for enhanced bio-compatibility, 8,16,29 surface patterning, 35,57 and for drug delivery. 40 Compared to the assembly of a single collagen type, co-assembly of multiple types is far less understood.…”

“…For studying initially formed fibrils or protofibrils, atomic force microscopy (AFM) is an effective approach. 8,9,23,30 AFM has been used for assembly of different collagen types including type-I, 5,13,17,21,23,30,37,52 type-II, 10 and type-V, 42 and also to visualize mature heterotypic fibrils including type-I/III 1 and type-I/V. 42 However, to our knowledge, the formation of heterotypic fibrils has not been analyzed previously at the level of individual fibrils.…”