3D Printed Engineered Nano-Composite Templates for Bone Regeneration

The overarching objective of the “3DPRENT? project is to facilitate translation of the 3D printing technology for bone regeneration. Three-dimensional (3D) bioprinting is seen as a potential new solution to create personalized bone-like constructs. However, lack of ideal bioinks is a considerable issue in 3D bioprinting, as various requirements related to cell function and printability of the bioinks exist. Tissue-specific hydrogels made from different formulations of natural polymers have been developed by the team. Our efforts indicated that standardized methods and protocols are required in the field of bioprinting for precise evaluation of growth and functionality of cells embedded in hydrogels. Biomaterial inks based on wood-derived cellulose nanofibrils (CNF) were developed and characterized for their material properties in terms of rheology, stability, and printability. Then, human bone marrow derived mesenchymal stem cells (MSC) were bioprinted with CNF as a bioink to optimize the crosslinking conditions in terms of cell viability and proliferation. The cytotoxicity of the CNF hydrogels and bioprinting process was evaluated. Furthermore, freeze-dried porous scaffolds were prepared using nano hydroxyapatite (nHA) and CNF. The results imply that CNF scaffolds with 20 % nHA has the greatest potential for further biomimetic bone tissue engineering investigations. The data underscore the significance of carefully evaluating the crosslinking strategy used in conjunction with hydrogels to achieve a balanced physical and biological property as well as a less complicated post-bioprinting procedure. Furthermore, a novel bioink (vascu-ink) for vascularized bone tissue has been developed. The ink was investigated for angiogenesis in different co-culture set-ups combining human umbilical vein endothelial cells (HUVECs) and human bone marrow stromal cells (hBMSCs). In addition to the high viability of the two cell types and significant scaffold colonization, tubular formation was recorded for both mixed-cell and separate-filaments culture model. The migration of hBMSCs, the organization of the HUVECs, and proliferation of both cell types was enabled by the bioink and the optimized cell type ratio. These results were confirmed with immunofluorescent staining and 3D confocal imaging of known angiogenic marker proteins. This data has also been under processing to quantify the volume, size and functionality of the tubular network to investigate clinical relevance. Moreover, initial results indicate that the mixed cell culture model has potential not only to initiate tubular formation but also osteogenic potential by demonstrating ECM maturation under osteogenic stimulation.

Recent clinical studies performed by our group and other clinical partners in Europe, propose alternatives to conventional treatment modalities by using the concept of tissue engineering in which engineered biomaterials (scaffolds) are used to deliver mesenchymal stem cells (MSC) and/or growth factors. Although there have been some successes, bone tissue engineering needs to overcome several challenges to meet clinical and commercialization needs. Among these challenges, the limitations of scaffolding biomaterials to mimic the macro to nanoscale structures of native tissues. Current bone scaffolds suffer from impaired cellular responses, inadequate delivery of growth factors, insufficient mechanical strength and incorrect design. The significance of the current project lies on combining nanotechnology and 3D printing technology. The synergetic impact of such integrated technologies has a potential to advance the field of bone tissue engineering by developing biomimetic multiscale multifunctional scaffolds for enhanced cell response and growth factor delivery. In 3DPRENT, oxidized cellulose nanofibers (CNF)-based hydrogel will be functionalized with nano-hydroxyapatite (nHA) for osteoconductivity, nanodiamond particles (nDP) to deliver vascular endothelial growth factor (VEGF) and finally to bioprint mesenchymal stem cells (MSC). For mechanical stability and vascularization, hydrogel layers will be reinforced with 3D printed microchannel network of a thermoplastic polymer modified with (nHA). The bioengineered constructs will be fabricated based on computational model-informed design, cultured in a dynamic in vitro conditions and finally validated in relevant pre-clinical animal models. 3DPRENT will develop not only outstanding basic scientific knowledge but also sustainable solutions and innovations based on nanotechnology, 3D printing and stem cells thus improving health and promoting new medical technology to meet the needs of society.