Structural Analysis of Reconstituted Collagen Type I - Heparin Cofibrils

Synthetic biomaterials are constantly being developed and play central roles in contemporary strategies in regenerative medicine and tissue engineering as artificial extracellular microenvironments. Such scaffolds provide 2D- and 3D-support for interaction with cells and thus convey spatial and temporal control over their function and multicellular processes, such as differentiation and morphogenesis. A model fibrillar system with tunable viscoelastic properties, comprised of 2 native ECM components like collagen type I and the GAG heparin, is presented here. Although the individual components comply with the adhesive, mechanical and bioinductive requirements for artificial reconstituted ECMs, their interaction and structural characterization remains an intriguing conundrum. The aim of the work was to analyze and structurally characterize a xenogeneic in vitro cell culture scaffold reconstituted from two native ECM components, collagen type I and the highly negatively charged glycosaminoglycan heparin. Utilizing a broad spectrum of structural analysis it could be shown that pepsin-solubilized collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape, with a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of GAG concentrations. The experiments imply a pronounced impact of the nucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with atelocollagen. Heparin is assumed to stabilize the collagen-GAG complexes and to enhance their parallel accretion during cofibrillogenesis, furthermore corroborated by the heparin quantitation data showing the GAG to be intercalated as a linker molecule with a specific binding site inside the cofibrils. In addition, the exerted morphogenic effect of the GAG, appears to be influenced by factors as degree of sulfation, charge, and concentration. Further detailed structural analysis of the PSC-heparin gels using TEM and SFM showed a hierarchy involving 3 different structural levels and banding patterns in the system: asymmetric segment longspacing (SLS) fibrils and symmetric segments with an average periodicity (AP) of 250 - 260 nm, symmetric fibrous longspacing (FLS IV) nanofibrils with AP of 165 nm, and cofibrils exhibiting an asymmetric D-periodicity of 67 nm with a striking resemblance to the native collagen type I banding pattern. The intercalation of the high negatively charged heparin in the cofibrils was suggested as the main trigger for the hierarchical formation of the polymorphic structures. We also proposed a model explaining the unexpected presence of a symmetric and asymmetric form in the system and the principles governing the symmetric or asymmetric fate of the molecules. The last section of the experiments showed that the presence of telopeptides and heparin both had significant effects on the structural and mechanical characteristics of in vitro reconstituted fibrillar collagen type I. The implemented structural analysis showed that the presence of telopeptides in acid soluble collagen (ASC) impeded the reconstitution of D-periodic collagen fibrils in the presence of heparin, leaving behind only a symmetric polymorphic form with a repeating unit of 165 nm (FLS IV). Further x-ray diffraction analysis of both telopeptide-free and telopeptide-intact collagen fibrils showed that the absence of the flanking non-helical termini in pepsin-solubilized collagen (PSC) resulted in a less compact packing of triple helices of atelocollagen with an increase of interhelical distance from 1.0 to 1.2 nm in dried samples. The looser packing of the triple helices was accompanied by a decrease in bending stiffness of the collagen fibrils, which demonstrated that the intercalated heparin cannot compensate for the depletion of telopeptides. Based on morphological, structural and mechanical differences between ASC and PSC-heparin fibrils reported here, we endorsed the idea that heparin acts as an intrafibrillar cross-linker which competed for binding sites at places along the atelocollagen helix that are occupied in vivo by telopeptides in the fibrillar collagen type I. The performed studies are of particular interest for understanding and gaining control over a rather versatile and already exploited xenogeneic cell culture system. The reconstituted cofibrils with their unusual morphology and GAG intercalation – a phenomenon not reported in vivo – are expected to exhibit interesting biochemical behavior as a biomaterial for ECM scaffolds. Varying the experimental conditions, extent of telopeptide removal, and heparin concentration provides powerful means to control the kinetics, structure, dimensions, as well as mechanical properties of the system which is particularly important for predicting a certain cell behavior towards the newly developed matrix. The GAG intercalation could be interesting for studies with required long-term 'release upon demand' of the GAG, as well as native binding and stabilization of growth factors, cytokines, chemokines, thus providing a secondary tool to control cell signaling and fate, and later on tissue morphogenesis.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Leibniz Institute of Polymer Research Dresden

Year

2010

Author dc:creator

• Stamov, Dimitar

Contributors dc:contributor

• Pompe, Tilo
• Müller, Daniel
• Werner, Carsten

• Biomaterials
• Collagen Type I
• Telopeptides
• Heparin
• Glycosaminoglycan
• GAG
• Extracellular Matrix
• ECM
• Structural Polymorphism
• Electron Microscopy
• Scanning Force Microscopy
• SFM
• Biomaterialien
• Kollagen Typ I
• Telopeptide
• Glykosaminoglykan
• Extrazelluläre Matrix
• Struktureller Polymorphismus
• Elektronenmikroskopie
• Rasterkraftmikroskopie