Endothelial of hybrid bioinkrevealed enhanced cell viability, proliferation, differentiationof

Endothelial progenitor cells (EPCs) area promising cell source for the treatmentof several ischemic diseases fortheir potentials in neovascularization.

1Nevertheless, the hostile ischemic conditions(e.g., low nutrition, oxidativestress, inflammation, reactive oxygenspecies) limit survival and differentiationof transplanted EPCs, and thus result inlow therapeutic efficacy.2 Atorvastatin, ahydroxymethylglutaryl-coenzyme A reductaseinhibitor, has exhibited good abilitiesto mobilize EPCs,3 enhances angiogenesisby reducing subdural hematoma,4and promotes endothelial cells function.5However, an efficient method for couplingthe therapeutic effects of atorvastatin andEPCs is still unmet.Tissue engineering can provide solutionssince it converges the usage of cells, biomaterials,and biological factors to restoreor replace the disorders. In particular, 3D cell printing is regarded as a versatile technique for tissueengineering due to the high freedom for positioning cells andbiomolecules in a wide range of biomaterials with predesignedpatterns and geometries.6 Particularly, 3D coaxial cell printinghas shown potentials for vascular tissue engineering becauseof the ability of directly fabricating perfusable vessel-mimickingstructure by extruding cell-laden biomaterials througha coupled core/shell nozzle.

7 In this technique, one indispensablerequirement for biomaterial is instant gelation behavior.Therefore, alginate-based hydrogel is widely used due to therapid ionic crosslinking via calcic treatment.7,8 However, thedeficiency of binding sites for cell attachment and migration inalginate drastically impairs activities of entrapped cells. Hence,it is necessary to seek an endothelial-inspiring material for thisengineering technique.Tissue-specific decellularized extracellular matrices (dECMs)have demonstrated the superiorities in mediating cellular functionscompared with other prevalent biomaterials.

9 Because, theycan uniquely recapitulate the inherent microenvironments of originaltissue including composition, structure, and biomechanical properties, which are critical regulators of cell fates such as survival,maturation, differentiation, and migration. In our previousstudies, a variety of tissue derived dECMs (e.g.

, adipose, cartilage,cardiac, muscle, and liver) have been successfully formulatedas printable bioink.10 Combining with 3D cell printing techniqueto modulate cell alignment and control graft structure,the dECM-based tissue analogues have achieved promoted cellactivities, enhanced tissue functions, and accelerated therapeuticeffects.10b,d Therefore, we believe that a vascular-tissue-deriveddECM (VdECM) bioink can compensate the drawback of alginateto both enable the direct tube printing and improve cell functions,and thus helps to produce biofunctional structure.In this study, we engineered a EPC/atorvastatin-loadedpoly(lactic-co-glycolic) acid (PLGA) microspheres (APMS) ladenbio-blood-vessel (BBV) by combining 3D coaxial cell printingtechnique and a hybrid bioink composed of VdECM and alginate(Figure 1A,B). The in vitro evaluations of hybrid bioinkrevealed enhanced cell viability, proliferation, differentiationof EPCs compared with type-I collagen. In addition, the hybridbioink enabled a direct fabrication of cell/drug laden tubes with varied dimensions by modulating the printing parameters.

Moreover, the observed in vitro endothelialization of perfusableBBV demonstrated its significant potentials to developfunctional blood vessel graft. The therapeutic efficacy of BBVwas investigated in a nude mice hind limb ischemia model(Figure 1C). The significantly improved performance of EPCsand promoted recovery rate of ischemic injury proved the superiortherapeutic effects of such a BBV-based method comparedto conventional cell and drug treatments.