Analysis of the Cellular Infiltration of Benzyl-Esterified Hyaluronan Sponges Implanted in Rats

Unseeded sponges of benzyl-esterified hyaluronan (HYAFF11) and HYAFF11 coated with unmodified hyaluronan were implanted subcutaneously and intramuscularly in adult rats for 1, 2, 4, 8, 12, and 26 weeks. Explanted samples were stained tincturally using Van Geison, von Kossa, and hematoxylin and eosin, enzyme histochemically by chloroacetate esterase, and by immunohistochemistry for the specific identification of cell types and subpopulations, targeting immature (ED1) and mature macrophages (ED2), MHC-I subset, MHC-II subset, CD54, T-cell alpha-beta receptor, T-cell gamma-delta receptor, CD2, CD4, CD8, natural killer cells, B-cells, vimentin, and TGFbeta. Little or no fibrous tissue formation was observed in any sample in either sponge type at any implantation site. Little degradation was observed in either location until 26 weeks. Little neovascularization occurred at early time periods but was in evidence at 26 weeks. Complete cellular infiltration was observed after 4 weeks, with some mature adipocytes observed within the center of the subcutaneous implants, but these cells were mainly observed around the periphery of the sponges. At 26 weeks, cells were mostly macrophages, with small numbers of T-lymphocytes present. No natural killer cells, B-cells, helper/inducer, or cytotoxic/suppressor T-cells were observed in any sample. Most infiltrating cells were MHC-II positive, and discrete pockets of TGFbeta protein were observed within the sponges. While a sustained inflammatory response was observed within both sponge types at 26 weeks, it was relatively benign and nonspecific immunologically, and inflammatory markers such as MHC-II were declining after 12 weeks. No fibrous capsule was observed, and sponge degradation was only observed at 26 weeks, an event essential for induction of neovasculargenesis. At 26 weeks, there was significant staining for vimentin and ED2 on macrophages. Taken with the pattern of other macrophage activation markers, angiogenic environment and absence of inhibitory matrix proteins, the conditions were consistent with the onset of neoadipogenesis, although this would need to be confirmed by longer term studies. For the generation of neoadipose tissue for clinical therapy, we hypothesize that macrophages require an inflammatory stimulus for infiltration, then a reduction in proinflammatory cytokine secretion simultaneous with angiogenic conditions allowing macrophage differentiation into adipocytes.