In situ bioprinting of high-density cell-laden core-shell microgel bioinks enabling cellular condensation for vascularized bone tissue regeneration

Abstract Bone damage and loss resulting from combat associated penetrating fragment projectiles, gunshot wounds and improvised explosive devices are some of the most common injuries US Military personnel experience and contribute to increasing Veterans’ health care costs. Yet, there are few viable options to consistently regenerate functional craniofacial bone for soldiers and veterans. Therefore, there is a significant need for the development of innovative therapeutic approaches to address this problem. Cell-laden biomaterial scaffold-based strategies face several challenges, such as limited cell-cell interactions, potential immune and/or inflammatory reaction, and unsynchronized scaffold degradation rate with new tissue formation. Promising new scaffold-free cellular condensation strategies can address these issues. To date, however, their formation has still required in vitro culture prior to implantation. To overcome this issue, we have engineered a new technology: immediately implantable, biodegradable and photocrosslinkable high-density hMSC-laden core-shell microgels enabling cell condensations and subsequent generation of functional tissues in vivo without in vitro culture. Using this system and 3D bioprinting, it is now possible to precisely engineer the architecture of osteogenic cell condensations for bone regeneration to match patient-specific defects. Recently, we have also engineered technology capable of 3D printing an individual cell-only bioink and maintaining the printed structures. By using the in situ bioprinted osteogenic core-shell microparticles as a support slurry bath, it is now possible to print defined 3D patterns of prevascular individual cell-only bioink into the slurry to generate a tissue construct composed of osteogenic hMSC condensations with an incorporated spatially patterned prevascular network of cell condensations. The rapid degradation of the microgel hydrogel shell layer will enable fusion of the osteogenic condensations with each other, the forming prevascular network and the surrounding host tissue. Locally delivered growth factor from incorporated microparticles will drive the local formation of the two different tissue types to promote the growth of functional vascularized bone tissue. We hypothesize that multi-tissue cell condensations can be fabricated directly in vivo in complex architectures using osteogenic core-shell microgel and individual cell-based prevasculogenic bioinks and 3D bioprinting technology to form patterned prevascularized bone constructs for healing critical-sized cranial defects without in vitro processing and culturing. Specifically, we aim to (1) examine the role of physical properties of the core-shell microgel bioink and printing parameters on the resolution and shape fidelity of the 3D bioprinted constructs, (2) engineer and evaluate 3D bioprinted prevasculature patterned high cell-density bone constructs, and (3) determine the preclinical potential of in situ 3D bioprinted prevasculature patterned osteogenic cell condensation constructs to heal critical-sized rat calvarial bone defects. This platform strategy has the potential to greatly enhance the lives of military personnel and veterans suffering from bone defects and may enable the engineering of other complex tissues in the body.