For collagen based tissue engineering it is important to know how the changes in water-biomolecules interactions are handled by the degree of cross-links in collagen nano-scaffolds. When hydration properties of collagens with different cross-linking are detailed by NMR a role of water interactions in improving collagen scaffold characteristics for tissue engineering could be clarified. A description of water diffusion in collagen fibres with cross-links should lead to better understanding of internal interactions and knowledge of porous media structure of collagen tissues. This paper presents the results of one- and two-dimensional NMR methods in studying molecular anisotropy and microstructure of collagen fibres from 2 connective tissues with different cross-linking levels (fibres from adult steer and young calf) at water content of 0.6 g water/g of dry matter. The NMR relaxation times (T1 and T2) have been studied in oriented collagen fibres (at 0° to the static magnetic field B0). The apparent translation diffusion coefficients (Dapp) at two directions of applied gradient – along the direction of static magnetic field B0 and perpendicular to B0 – have been studied in oriented collagen fibres (at 0° to the static magnetic field B0). The diffusion time dependence of Dapp showed for both directions of applied gradient a restriction for motion of water molecules. A model of equally spaced plane parallel permeable barriers developed by Tanner for NMR diffusion studies has been used for the estimation of restricted distance and permeability coefficient. Anisotropy of Dapp in oriented fibres was observed in both collagen tissues. Application of 2D DDCOSY NMR spectroscopy with two pairs of collinear gradients on collagen fibres oriented along the static magnetic field and simulations of diffusion anisotropy showed similar features on 2D diffusion maps.