Genetically-programmed, mesenchymal stromal cell-laden & mechanically strong 3D bioprinted scaffolds for bone repair

Abstract

Additive manufacturing processes used to create regenerative bone tissue engineered implants are not biocompatible, thereby restricting direct use with stem cells and usually require cell seeding post-fabrication. Combined delivery of stem cells with the controlled release of osteogenic factors, within a mechanically-strong biomaterial combined during manufacturing would replace injectable defect fillers (cements) and allow personalized implants to be rapidly prototyped by 3D bioprinting. Through the use of direct genetic programming via the sustained release of an exogenously delivered transcription factor RUNX2 (delivered as recombinant GET-RUNX2 protein) encapsulated in PLGA microparticles (MPs) , we demonstrate that human mesenchymal stromal (stem) cells (hMSCs) can be directly fabricated into a thermo-sintered 3D bioprintable material and achieve effective osteogenic differentiation. Importantly we observed osteogenic programming of gene expression by released GET-RUNX2 (8.2-, 3.3- and 3.9-fold increases in OSX, RUNX2 and OPN expression, respectively) and calcification (von Kossa staining) in our scaffolds. The developed biodegradable PLGA/PEG paste formulation augments high-density bone development in a defect model (~2.4-fold increase in high density bone volume) and can be used to rapidly prototype clinically-sized hMSC-laden implants within minutes using mild, cytocompatible extrusion bioprinting. The ability to create mechanically strong 'cancellous bone-like’ printable implants for tissue repair that contain stem cells and controlled-release of programming factors is innovative, and will facilitate the development of novel localized delivery approaches to direct cellular behaviour for many regenerative medicine applications including those for personalized bone repair.

Bioengineered Living Bone Grafts—A Concise Review on Bioreactors and Production Techniques In Vitro

“…They showed that the developed novel scaffolds increased the osteogenic differentiation of encapsulated cells and ECM mineralization. Awwad et al [60] bioprinted PLGA/PEGbased bone scaffolds comprised of the active GET (glycosaminoglycan-binding enhanced transduction system)-RUNX2 protein and human MSCs. Bioprinted scaffolds ensured controlled release of transcription factors, affecting the osteogenic differentiation of the cells.…”

3D Printed Bioconstructs: Regenerative Modulation for Genetic Expression

“…The in-vitro studies demonstrated that the seeded stem cells induced higher osteogenesis action on exposure to the transcription factor due to the sustained release profile of RUNX2 from the encapsulated microparticles. [40] On the other hand, 3D printed scaffolds are also capable in modulating gene expression as proved by an experiment, wherein hydrogels were developed for regeneration of bone tissue with the help of biocompatible bioinks, namely gelatin and alginate. The conformational changes in the fabricated scaffolds such as size, porosity and mechanical properties altered the osteogenic gene expression of MC3T3-E1 cells.…”

Comprehensive Review on Design and Manufacturing of Bio-scaffolds for Bone Reconstruction

“…Polypropylene fumarate (PPF), free radical polymerized poly­(ethylene glycol)-polycaprolactone (PEG-PCL-PEG), and pluronic (PF 127) combined with Simvastatin was employed in forming a bio-ink used in the formation of pseudo bone scaffold to promote healing and repair of the bone defect . The formation of bone scaffolds with genetically modified protein molecules along with mesenchymal stromal cells was attempted, where the importance of temperature-responsive polymeric microparticles such as PEG could ideally solidify under human body temperature formatting a cancellous bone-like structure has been stated . Heterogeneous tissue constructs containing a bone and cartilage matrix were developed using alginate alone and alginate supplemented with biphasic calcium phosphate, hydroxyapatite, and β-tricalcium phosphate for printing MSCs and chondrocytes.…”

“…211 The formation of bone scaffolds with genetically modified protein molecules along with mesenchymal stromal cells was attempted, where the importance of temperature-responsive polymeric microparticles such as PEG could ideally solidify under human body temperature formatting a cancellous bone-like structure has been stated. 212 Heterogeneous tissue constructs containing a bone and cartilage matrix were developed using alginate alone and alginate supplemented with biphasic calcium phosphate, hydroxyapatite, and β-tricalcium phosphate for printing MSCs and chondrocytes.…”