We and others have shown that changing surface characteristics of the spacer implanted during the first Masquelet stage alters some aspects of membrane development. Previously we demonstrated that titanium spacers create membranes that are better barriers to movement of solutes >70kDa in size than polymethyl methacrylate (PMMA) induced-membranes, and roughening creates more mechanically compliant membranes. However, it is unclear if these alterations affect the membrane’s biochemical environment or bone regeneration during the second stage. Ten-week-old, male Sprague-Dawley rats underwent an initial surgery to create an externally stabilized 6mm femoral defect. PMMA or titanium spacers with smooth (~1um) or roughened (~8um) surfaces were implanted. Four weeks later, rats were either euthanized for membrane harvest or underwent the second Masquelet surgery. Titanium spacers induced thicker membranes that were similar in structure and biochemical expression. All membranes were bilayered with the inner layer having increased factor expression (BMP2, TGFβ, IL6, and VEGF). Roughening increased overall IL6 levels. Ten-weeks post-engraftment, PMMA-smooth induced membranes better supported bone regeneration (60% union). The other groups only had 1 or 2 that united (9–22%). There were no significant differences in any microCT or dynamic histology outcome. In conclusion, this study suggests that the membrane’s important function in the Masquelet technique is not simply as a barrier. There is likely a critical biochemical, cellular, or vascular component as well.