Recent advances in regenerative medicine have confirmed the potential to manufacture viable and effective tissue engineering 3D constructs comprising living cells for tissue repair and augmentation. Cell printing has shown promising potential in cell patterning in a number of studies enabling stem cells to be precisely deposited as a blueprint for tissue regeneration guidance. Such manufacturing techniques, however, face a number of challenges including; (i) post-printing cell damage, (ii) proliferation impairment and, (iii) poor or excessive final cell density deposition. The use of hydrogels offers one approach to address these issues given the ability to tune these biomaterials and subsequent application as vectors capable of delivering cell populations and as extrusion pastes. While stem cell-laden hydrogel 3D constructs have been widely established in vitro , clinical relevance, evidenced by in vivo long-term efficacy and clinical application, remains to be demonstrated. This review explores the central features of cell printing, cell-hydrogel properties and cell-biomaterial interactions together with the current advances and challenges in stem cell printing. A key focus is the translational hurdles to clinical application and how in vivo research can reshape and inform cell printing applications for an ageing population.

Bioprinting of living cells is rapidly developing as an advanced biofabrication approach to engineer tissues. Bioinks can be extruded in three-dimensions (3D) to fabricate complex and hierarchical constructs for implantation. However, lack of functionality can often be attributed to poor bioink properties. Indeed, advanced bioinks encapsulating living cells should: (i) present optimal rheological properties and retain 3D structure post-fabrication, (ii) promote cell viability and support cell differentiation, (iii) localise proteins of interest (e.g. vascular endothelial growth factor (VEGF)) to stimulate encapsulated cell activity and tissue ingrowth upon implantation. In this study, we present the results of the inclusion of a synthetic nanoclay, Laponite (LPN) together with a gelatin methacryloyl (GelMA) bioink and the development of a functional cellinstructive bioink. A nanocomposite bioink displaying enhanced shape fidelity retention and interconnected porosity within extrusion-bioprinted fibres was observed. Human bone marrow stromal cell (HBMSC) viability within the nanocomposite showed no significant changes over 21 days of culture in LPN-GelMA (85.60 ± 10.27 %), compared to a significant decrease in GelMA from 7 (95.88 ± 2.90 %) to 21 days (55.54 ± 14.72 %) (p<0.01). HBMSCs were observed to proliferate in LPN-GelMA with a significant increase in cell number over 21 days (p<0.0001) compared to GelMA alone. HBMSCsladen LPN-GelMA scaffolds supported osteogenic differentiation evidenced by mineralized nodule formation, including in the absence of the osteogenic drug dexamethasone. Ex vivo implantation in a chick chorioallantoic membrane (CAM) model, demonstrated excellent integration of the bioink constructs in the vascular chick embryo after 7 days of incubation. VEGF-loaded LPN-GelMA constructs demonstrated significantly higher vessel penetration than GelMA-VEGF (p<0.0001) scaffolds.Integration and vascularisation was directly related to increased drug absorption and retention by LPN-GelMA compared to LPN-free GelMA. In summary, a novel lightcurable nanocomposite bioink for 3D skeletal regeneration supportive of cell growth and growth factor retention and delivery, evidenced by ex vivo vasculogenesis, was developed with potential application in hard and soft tissue reparation.

Neuronal function critically depends on coordinated subcellular distribution of mRNAs. Disturbed mRNA processing and axonal transport has been found in spinal muscular atrophy and could be causative for dysfunction and degeneration of motoneurons. Despite the advances made in characterizing the transport mechanisms of several axonal mRNAs, an unbiased approach to identify the axonal repertoire of mRNAs in healthy and degenerating motoneurons has been lacking. Here we used compartmentalized microfluidic chambers to investigate the somatodendritic and axonal mRNA content of cultured motoneurons by microarray analysis. In axons, transcripts related to protein synthesis and energy production were enriched relative to the somatodendritic compartment. Knockdown of Smn, the protein deficient in spinal muscular atrophy, produced a large number of transcript alterations in both compartments. Transcripts related to immune functions, including MHC class I genes, and with roles in RNA splicing were up-regulated in the somatodendritic compartment. On the axonal side, transcripts associated with axon growth and synaptic activity were down-regulated. These alterations provide evidence that subcellular localization of transcripts with axonal functions as well as regulation of specific transcripts with nonautonomous functions is disturbed in Smn-deficient motoneurons, most likely contributing to the pathophysiology of spinal muscular atrophy.

Axonal transport and translation of beta-actin mRNA plays an important role for axonal growth and presynaptic differentiation in many neurons including hippocampal, cortical and spinal motor neurons. Several beta-actin mRNA-binding and transport proteins have been identified, including ZBP1, ZBP2 and hnRNP-R. hnRNP-R has been found as an interaction partner of the survival motor neuron protein that is deficient in spinal muscular atrophy. Little is known about the function of hnRNP-R in axonal beta-actin translocation. hnRNP-R and beta-actin mRNA are colocalized in axons. Recombinant hnRNP-R interacts directly with the 3'-UTR of beta-actin mRNA. We studied the role of hnRNP-R in motor neurons by knockdown in zebrafish embryos and isolated mouse motor neurons. Suppression of hnRNP-R in developing zebrafish embryos results in reduced axon growth in spinal motor neurons, without any alteration in motor neuron survival. ShRNA-mediated knockdown in isolated embryonic mouse motor neurons reduces beta-actin mRNA translocation to the axonal growth cone, which is paralleled by reduced axon elongation. Dendrite growth and neuronal survival were not affected by hnRNP-R depletion in these neurons. The loss of beta-actin mRNA in axonal growth cones of hnRNP-R-depleted motor neurons resembles that observed in Smn-deficient motor neurons, a model for the human disease spinal muscular atrophy. In particular, hnRNP-R-depleted motor neurons also exhibit defects in presynaptic clustering of voltage-gated calcium channels. Our data suggest that hnRNP-R-mediated axonal beta-actin mRNA translocation plays an essential physiological role for axon growth and presynaptic differentiation.

Acellular soft hydrogels are not ideal for hard tissue engineering given their poor mechanical stability, however, in combination with cellular components offer significant promise for tissue regeneration. Indeed, nanocomposite bioinks provide an attractive platform to deliver human bone marrow stromal cells (HBMSCs) in three dimensions producing cellladen constructs that aim to facilitate bone repair and functionality. Here we present the in vitro, ex vivo and in vivo investigation of bioprinted HBMSCs encapsulated in a nanoclaybased bioink to produce viable and functional three-dimensional constructs. HBMSC-laden constructs remained viable over 21 days in vitro and immediately functional when conditioned with osteogenic media. 3D scaffolds seeded with human umbilical vein endothelial cells (HUVECs) and loaded with vascular endothelial growth factor (VEGF) implanted ex vivo into a chick chorioallantoic membrane (CAM) model showed integration and vascularisation after 7 days of incubation. In a pre-clinical in vivo application of a nanoclay-based bioink to regenerate skeletal tissue, we demonstrated bone morphogenetic protein-2 (BMP-2) absorbed scaffolds produced extensive mineralisation after 4 weeks (p<0.0001) compared to the drug-free and alginate controls. In addition, HBMSC-laden 3D printed scaffolds were found to significantly (p<0.0001) support bone tissue formation in vivo compared to acellular and cast scaffolds. These studies illustrate the potential of nanoclay-based bioink, to produce viable and functional constructs for clinically relevant skeletal tissue regeneration.