To examine biocompatibility in vivo

To examine biocompatibility in vivo, we subcutaneously implanted HAPLGA scaffolds (that is, HB) in female BALB/c mice. HA/PLGA (1:1) hot-melt scaffolds were also implanted as a comparison, because they are similar to materials that have been previously used and eval- uated as bone implants (32). After 7 days, tissue had already begun to infiltrate and vascularize throughout the HB scaffolds (fig. S7A). The hot-melt scaffold explants could not be histologically processed suc- cessfully, a result of their highly brittle nature and the dissolution of the PLGA (majority scaffold component) in histological process solvents,hich caused the loss of integrated tissues and embedded HA particles during processing (Fig. 6F). After 35 days in vivo, the HB scaffolds were completely integrated with the surrounding host tissue (Fig. 6, A to C). SEM imaging of explanted scaffold tissues revealed that, in both material systems, the tissue formed intimate contact with the material within and throughout the scaffold volume by day 35. However, there was a distinct difference in the structure and texture of the tissue within HB (Fig. 6, D and E) compared to that within the hot-melt printed scaffolds (Fig. 6, G and H). Tissue surrounding HB more closely mimicked healthy ECM, with defined collagenous ECM (fig. S8) and blood vessels ranging from 2-mm single-cell capillaries to multihundred-micrometer vessels present throughout the scaffold (Fig. 6, B to D, and fig. S8). In contrast, the tissue within the 1:1 HA/PLGA hot-melt scaffolds was characteristically dense and relatively acellular compared to the HB counterparts (Fig. 6, G and H). SEM imaging also revealed a population of unhealthy blood cells (burr cells) inhabiting the tissue within the 1:1 HA/PLGA scaffolds (Fig. 6I), which may indicate a strong local fibrotic response. Additional staining with Alizarin Red S did not indicate any obvious mineralization within the integrated tissues by day 35 (fig. S7C)