Kinetics of <i>De Novo</i> Bone and Bone Marrow Niche Formation With Hybrid Click Cryogels

Compromised bone marrow niches following irradiation limit hematopoietic stem cell transplantation (HSCT) success by delaying immune reconstitution. Strategies to rebuild functional marrow environments are essential to support hematopoietic stem cell (HSC) maintenance. However, the fundamental relationship between bone formation and bone marrow niche development, and how these processes are modulated by key biological variables, remains poorly understood. Here, we present an alginate-gelatin cryogel platform that provides sustained BMP-2 release for ectopic osteogenesis and use it to systematically investigate the effects of BMP-2 dose, host immune status, and biological sex. Within 2-4 weeks, the cryogels supported the formation of a cortical bone shell and an internal trabecular bone network populated by hematopoietic tissue. Increasing the BMP-2 dose accelerated mineralization and doubled the resident HSC population, whereas immunocompromised hosts displayed a two-week delay in niche development and a fourfold reduction in HSCs. Female mice exhibited enhanced niche formation relative to males under identical conditions. These findings establish BMP-2-releasing hybrid click cryogels as a tunable bone marrow niche platform and highlight how host- and dose-dependent parameters modulate engineered niche formation, providing insights that may inform future strategies to improve immune reconstitution following HSCT.