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Calcium and integrin-binding protein 2 (CIB2) belongs to a protein family with four known members, CIB1 through CIB4, which are characterized by multiple calcium-binding EF-hand domains. Among the family members, the Cib1 and Cib2 genes are expressed in mouse cochlear hair cells, and mutations in the human CIB2 gene have been associated with nonsyndromic deafness DFNB48 and syndromic deafness USH1J. To further explore the function of CIB1 and CIB2 in hearing, we established Cib1 and Cib2 knockout mice using the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease (CRISPR/Cas9) genome editing technique. We found that loss of CIB1 protein does not affect auditory function, whereas loss of CIB2 protein causes profound hearing loss in mice. Further investigation revealed that hair cell stereocilia development is affected in Cib2 knockout mice. Noticeably, loss of CIB2 abolishes mechanoelectrical transduction (MET) currents in auditory hair cells. In conclusion, we show here that although both CIB1 and CIB2 are readily detected in the cochlea, only loss of CIB2 results in profound hearing loss, and that CIB2 is essential for auditory hair cell MET.

Vascularization and bone regeneration are two closely related processes during bone reconstruction. A three-dimensional (3D) scaffold with porous architecture provides a suitable microenvironment for vascular growth and bone formation. Here, we present a simple and general strategy to construct a nanofibrous poly( l -lactide)/poly(ε-caprolactone) (PLLA/PCL) scaffold with interconnected perfusable microchannel networks (IPMs) based on 3D printing technology by combining the phase separation and sacrificial template methods. The regular and customizable microchannel patterns within the scaffolds (spacings: 0.4 mm, 0.5 mm, and 0.6 mm; diameters: 0.8 mm, 1 mm, and 1.2 mm) were made to investigate the effect of microchannel structure on angiogenesis and osteogenesis. The results of subcutaneous embedding experiment showed that 0.5/0.8-IPMs (spacing/diameter = 0.5/0.8) and 0.5/1-IPMs (spacing/diameter = 0.5/1) scaffolds exhibited more vascular network formation as compared with other counterparts. After loading with vascular endothelial growth factor (VEGF), VEGF@IPMs-0.5/0.8 scaffold prompted better human umbilical vein endothelial cells (HUVECs) migration and neo-blood vessel formation, as determined by Transwell migration, scratch wound healing, and chorioallantoic membrane (CAM) assays. Furthermore, the microangiography and rat cranial bone defects experiments demonstrated that VEGF@IPMs-0.5/0.8 scaffold exhibited better performance in vascular network formation and new bone formation compared to VEGF@IPMs-0.5/1 scaffold. In summary, our results suggested that the microchannel structure within the scaffolds could be tailored by an adjustable caramel-based template strategy, and the combination of interconnected perfusion microchannel networks and angiogenic factors could significantly enhance vascularization and bone regeneration.

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Three-dimensional (3D) printed scaffolds provide promising perspective in bone tissue engineering. 3D printed scaffolds with micro- and nano-fibrous structure that facilitates the cell adhesion and migration, and combined vascularization and...

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IntroductionThe fast degradation of vascular graft and the infiltration of smooth muscle cells (SMCs) into the vascular graft are considered to be critical for the regeneration of functional neo-vessels. In our previous study, a novel dual phase separation technique was developed to one-pot prepare macroporous nanofibrous poly(L-lactic acid) (PLLA)/poly(ε-caprolactone) (PCL) vascular scaffold by phase separating the immiscible polymer blend. However, the slow degradation of PLLA/PCL limited cell infiltration. Herein, we hypothesized that poly(lactic-co-glycolic acid) (PLGA) would be miscible with PLLA but immiscible with PCL. Then, PLGA can be introduced into the PLLA/PCL blend to fabricate macroporous nanofibrous scaffold with improved biodegradability by using dual phase separation technique.Materials and methodsThe miscibility of PLGA with PLLA and PCL was evaluated. Then, the PLLA/PLGA/PCL scaffold was prepared by dual phase separation technique. The prepared scaffolds were characterized in terms of the morphology, in vitro degradation, mechanical properties, and cells’ infiltration and viability for human vascular SMCs (HVSMCs). Finally, platelet-derived growth factor-BB (PDGF-BB) was immobilized on the scaffold and its effect on the bioactivity of HVSMCs was studied.ResultsPLGA is miscible with PLLA but immiscible with PCL as hypothesized. The addition of PLGA enlarged the pore size and improved the biodegradability of composite scaffold. Notably, PLLA/PLGA/PCL scaffold with the blend ratio of 30:40:30 possessed improved pore interconnectivity for cells’ infiltration and enough mechanical properties. Moreover, HVSMCs could grow and infiltrate into this scaffold, and surface modification with PDGF-BB on the nanofibrous scaffold enhanced HVSMCs migration and proliferation.ConclusionThis study provides a strategy to expand dual phase separation technique into utilizing ternary even multinary polymer blend to fabricate macroporous nanofibrous scaffold with improved physicochemical properties. The prepared PLLA/PLGA/PCL scaffold would be promising for the regeneration of functional tunica media in vascular tissue engineering.