Characterization of biomaterial-tissue interactions in their three-dimensional context - an in vivo study

摘要

Synthetic biomaterials are widely used for the treatment of chronic and acute wounds. They play an increasingly role in regenerative medicine. However, the adaptations and interactions of biological tissue at implantation site with biomaterial of complex architecture are difficult to characterize in their three-dimensional (3D) context. This morphometric study uses computerized quantification of synchrotron radiation x-ray tomographic microscopic (SRXTM) data to evaluate the interplay of a filamentous synthetic copolymer, mainly based on DL-lactic acid, with host tissue in pigs. Complementary analysis and cross-validation were performed by light- and electron microscopy. SRXTM allows for nondestructive 3D analysis of biomaterial on a sub-micron level. Thereby parameters defining filamentous biomaterial, such as filament-diameter, -volume-density, -porosity, -directionality and -clustering, are now available for quantification without stereological detriments (Fig.1,2B). At the same time, SRXTM provides a sufficient spatial resolution (voxel-edge-length: 325nm) to visualize and quantify single biomaterial-induced multinucleated giant cells (BIMGC) in their spatial extend and relation to the synthetic filaments (Fig.2B,3C/D). Cell and collagen fiber directionality are assessable in 3D and can be correlated to filament alignment (Fig.2B,3B). Scanning and transmission electron microscopy demonstrate organelle containing, ultra-fine, finger-like cell protrusions into the blind ends of the biomaterial filaments (Fig. 2C/D). One SRXTM-reconstruction of the materials prior to implantation, and 30 days after implantation were analyzed. The filaments were segmented in 3D using thresholding and suitable morphological operators. In the post-implantation volume image, BIMGCs have been identified based on their 3D-texture. BIMGCs can be identified with high sensitivity. Furthermore, cell and collagen fiber directionality have been quantified using the MAVI software package (Fraunhofer ITWM, Kaiserslautem). Results show that the pre- and post-implantation filament diameters remain unchanged (21.6±5.0μm vs. 23.3±6.9μm, respectively). However,, there is a dear densification of the fiber system (increasing from 9.5vol% pre to 19.6vol% post). The measured fiber volume densities VV give clear indication for 3D-fiber clustering post-implantation in terms of an increasing standard deviation of W (3.1vol% pre vs. 4.3vol% post). Regarding 3D-morphometry, BIMGCs were largely non-convex with volumes mostly below 60kμm~3 (Fig.3C/D). With the present study we demonstrate that SRXTM allows qualitative and quantitative 3D investigation of biomaterial-host-tissue interactions with sufficient spatial resolution to detect single cells. Cross-validation with histologic tissue samples and electron micrographs correlate well with synchrotron imaging results and provide complementary insights into interactive and adaptive processes.