Quantitative assessment of the tissue response to films of hyaluronan derivatives

The aim of this study was to evaluate the in vivo response following implantation into a rat model of three innovative hyaluronan derivatives for clinical use: HYAFF 7, HYAFF 11 and HYAFF 11p75 (respectively, the 100% ethyl ester, 100% and 75% benzyl esters). The tissue reaction evoked by films of these new biomaterials implanted into the dorsolumbar musculature of rats was assessed quantitatively using a well established technique based upon an image analysis system. The number of inflammatory cells present and the patterns of cell distribution around the implant up to a distance of 642 microns were examined at different time periods after implantation. Since a well-delineated tissue-material interface was needed for this type of investigation, it was not possible to apply image analysis to sections once dissolution of the implanted materials had begun. Films of both the total esters, HYAFF 7 and HYAFF 11, were found to undergo a slow dissolution process and, after a month, films of these materials were still present at the site of implantation. Differences in response to the two materials were observed only during the first two weeks, particularly with respect to neutrophil distribution and total cellularity. HYAFF 7 was found to be more reactive, with higher numbers of neutrophils near the surface of the implant than HYAFF 11. Thereafter, the differences between the two materials were minimal and owing mainly to a faster dissolution of HYAFF 7 films. After 3 and 5 months, considerable degradation of films of both total esters had occurred. Significant quantities of material appeared inside numerous macrophages with an ED1-positive phenotype. Only a very thin layer of fibrous connective tissue, indicative of low reactivity, was found to surround the site of implantation, separating the dissolved material and the phagocytic cells from healthy muscular tissue. ED2-positive macrophages were primarily confined within the lining connective tissue. The partial benzyl ester, HYAFF 11p75, showed a different behaviour. In fact, evidence of film dissolution was already present a week after the implantation. After two weeks, the implanted films were completely dissolved and numerous ED1-positive macrophages phagocytosing the material were observed at the site of implantation. Therefore, in agreement with previous in vitro studies, which showed a greater susceptibility to degradation of hyaluronan derivatives with lower percentage of esterification, HYAFF 11p75 underwent resorption faster than the corresponding total ester.