Development of an effective treatment to guide bone repair in critical size bone defect applications remains a major unmet challenge. Current clinical gold standards display significant disadvantages, thereby necessitating that research focus on designing and producing a suitable alternative for healing. In this study, we comprehensively assessed the bone regenerative potential of a newly formulated 6-Bromoindirubin-3'-Oxime (BIO) incorporated chitosan-based scaffold using a mouse femoral defect model. Live 3D in vivo micro-CT imaging enabled us to monitor the progression of bone formation over 56 days, without needing additional replicates. Results demonstrated smaller distances between bone ends (1.033 ± 0.512 mm) compared to controls (1.474 ± 0.465 mm) at later timepoints (p = 0.0430), suggesting improved bone formation. This observed effect was supported with serum procollagen type I N-propeptide levels, where BIO scaffolds showed marked increases in collagen synthesis. As vascularization is often-overlooked, blood vessel density at 56 days was also assessed, showing an additional benefit of BIO incorporated scaffolds (9.264 ± 0.578) over controls (6.667 ± 1.300) on angiogenesis. Although BIO's incorporation did not lead to bony bridging or a significant difference in bone volume compared to controls at day 56, our findings suggest the BIO incorporated scaffold's ability to improve healing outcomes through enhancement of Wnt signaling. Further studies aimed at optimizing the dose to target this pathway are warranted, as a means to more completely regenerate bone in challenging healing scenarios.