Controlled Co-delivery of pPDGF-B and pBMP-2 from intraoperatively bioprinted bone constructs improves the repair of calvarial defects in rats.

Intraoperative bioprinting (IOB), which refers to the bioprinting process performed on a live subject in a surgical setting, has made it feasible to directly deliver gene-activated matrices into craniomaxillofacial (CMF) defect sites. In this study, we demonstrated a novel approach to overcome the current limitations of traditionally fabricated non-viral gene delivery systems through direct IOB of bone constructs into defect sites. We used a controlled co-delivery release of growth factors from a gene-activated matrix (an osteogenic bioink loaded with plasmid-DNAs (pDNA)) to promote bone repair. The controlled co-delivery approach was achieved from the combination of platelet-derived growth factor-B encoded plasmid-DNA (pPDGF-B) and chitosan-nanoparticle encapsulating pDNA encoded with bone morphogenetic protein-2 (CS-NPs(pBMP2)), which facilitated a burst release of pPDGF-B in 10 days, and a sustained release of pBMP-2 for 5 weeks in vitro. The controlled co-delivery approach was tested for its potential to repair critical-sized rat calvarial defects. The controlled-released pDNAs from the intraoperatively bioprinted bone constructs resulted in ∼40% bone tissue formation and ∼90% bone coverage area at 6 weeks compared to ∼10% new bone tissue and ∼25% total bone coverage area in empty defects. The delivery of growth factors incorporated within the intraoperatively bioprinted constructs could pose as an effective way to enhance bone regeneration in patients with cranial injuries in the future.

Intra-Operative Bioprinting of Hard, Soft, and Hard/Soft Composite Tissues for Craniomaxillofacial Reconstruction.

Reconstruction of complex craniomaxillofacial (CMF) defects is challenging due to the highly organized layering of multiple tissue types. Such compartmentalization necessitates the precise and effective use of cells and other biologics to recapitulate the native tissue anatomy. In this study, intra-operative bioprinting (IOB) of different CMF tissues, including bone, skin, and composite (hard/soft) tissues, is demonstrated directly on rats in a surgical setting. A novel extrudable osteogenic hard tissue ink is introduced, which induced substantial bone regeneration, with ≈80% bone coverage area of calvarial defects in 6 weeks. Using droplet-based bioprinting, the soft tissue ink accelerated the reconstruction of full-thickness skin defects and facilitated up to 60% wound closure in 6 days. Most importantly, the use of a hybrid IOB approach is unveiled to reconstitute hard/soft composite tissues in a stratified arrangement with controlled spatial bioink deposition conforming the shape of a new composite defect model, which resulted in ≈80% skin wound closure in 10 days and 50% bone coverage area at Week 6. The presented approach will be absolutely unique in the clinical realm of CMF defects and will have a significant impact on translating bioprinting technologies into the clinic in the future.

Collagen-infilled 3D printed scaffolds loaded with miR-148b-transfected bone marrow stem cells improve calvarial bone regeneration in rats.

Differentiation of progenitors in a controlled environment improves the repair of critical-sized calvarial bone defects; however, integrating micro RNA (miRNA) therapy with 3D printed scaffolds still remains a challenge for craniofacial reconstruction. In this study, we aimed to engineer three-dimensional (3D) printed hybrid scaffolds as a new ex situ miR-148b expressing delivery system for osteogenic induction of rat bone marrow stem cells (rBMSCs) in vitro, and also in vivo in critical-sized rat calvarial defects. miR-148b-transfected rBMSCs underwent early differentiation in collagen-infilled 3D printed hybrid scaffolds, expressing significant levels of osteogenic markers compared to non-transfected rBMSCs, as confirmed by gene expression and immunohistochemical staining. Furthermore, after eight weeks of implantation, micro-computed tomography, histology and immunohistochemical staining results indicated that scaffolds loaded with miR-148b-transfected rBMSCs improved bone regeneration considerably compared to the scaffolds loaded with non-transfected rBMSCs and facilitated near-complete repair of critical-sized calvarial defects. In conclusion, our results demonstrate that collagen-infilled 3D printed scaffolds serve as an effective system for miRNA transfected progenitor cells, which has a promising potential for stimulating osteogenesis and calvarial bone repair.

Thermally-controlled extrusion-based bioprinting of collagen.

Thermally-crosslinked hydrogels in bioprinting have gained increasing attention due to their ability to undergo tunable crosslinking by modulating the temperature and time of crosslinking. In this paper, we present a new bioink composed of collagen type-I and Pluronic® F-127 hydrogels, which was bioprinted using a thermally-controlled bioprinting unit. Bioprintability and rheology of the composite bioink was studied in a thorough manner in order to determine the optimal bioprinting time and extrusion profile of the bioink for fabrication of three-dimensional (3D) constructs, respectively. It was observed that collagen fibers aligned themselves along the directions of the printed filaments after bioprinting based on the results on an anisotropy study. Furthermore, rat bone marrow-derived stem cells (rBMSCs) were bioprinted in order to determine the effect of thermally-controlled extrusion process. In vitro viability and proliferation study revealed that rBMSCs were able to maintain their viability after extrusion and attached to collagen fibers, spread and proliferated within the constructs up to seven days of culture.