Coaxial electrohydrodynamic printing of core–shell microfibrous scaffolds with layer-specific growth factors release for enthesis regeneration

The rotator cuff tear has emerged as a significant global health concern.However,existing therapies fail to fully restore the intricate bone-to-tendon gradients,resulting in compromised biomechanical functionalities of the reconstructed enthesis tissues.Herein,a tri-layered core–shell microfibrous scaffold with layer-specific growth factors(GFs)release is developed using coaxial electrohydrodynamic(EHD)printing for in situ cell recruitment and differentiation to facilitate gradient enthesis tissue repair.Stromal cell-derived factor-1(SDF-1)is loaded in the shell,while basic fibroblast GF,transforming GF-beta,and bone morphogenetic protein-2 are loaded in the core of the EHD-printed microfibrous scaffolds in a layer-specific manner.Correspondingly,the tri-layered microfibrous scaffolds have a core–shell fiber size of(25.7±5.1)μm,with a pore size sequentially increasing from(81.5±4.6)μm to(173.3±6.9)μm,and to(388.9±6.9μm)for the tenogenic,chondrogenic,and osteogenic instructive layers.A rapid release of embedded GFs is observed within the first 2 d,followed by a faster release of SDF-1 and a slightly slower release of differentiation GFs for approximately four weeks.The coaxial EHD-printed microfibrous scaffolds significantly promote stem cell recruitment and direct their differentiation toward tenocyte,chondrocyte,and osteocyte phenotypes in vitro.When implanted in vivo,the tri-layered core–shell microfibrous scaffolds rapidly restored the biomechanical functions and promoted enthesis tissue regeneration with native-like bone-to-tendon gradients.Our findings suggest that the microfibrous scaffolds with layer-specific GFs release may offer a promising clinical solution for enthesis regeneration.

Functionalized alginate-based bioinks for microscale electrohydrodynamic bioprinting of living tissue constructs with improved cellular spreading and alignment

Bioprinting has been widely investigated for tissue engineering and regenerative medicine applications.However,it is still difficult to reconstruct the complex native cell arrangement due to the limited printing resolution of conventional bioprinting techniques such as extrusion-and inkjet-based printing.Recently,an electrohydrodynamic(EHD)bioprinting strategy was reported for the precise deposition of well-organized cell-laden constructs with microscale filament size,whereas few studies have been devoted to developing bioinks that can be applied for EHD bioprinting and simultaneously support cell spreading.This study describes functionalized alginate-based bioinks for microscale EHD bioprinting using peptide grafting and fibrin incorporation,which leads to high cell viability(>90%)and cell spreading.The printed filaments can be further refined to as small as 30μm by incorporating polyoxyethylene and remained stable over one week when exposed to an aqueous environment.By utilizing the presented alginate-based bioinks,layer-specific cell alignment along the printing struts could be observed inside the EHD-printed microscale filaments,which allows fabricating living constructs with cell-scale filament resolution for guided cellular orientation.

FRUCTOKINASE-LIKE PROTEIN 1 interacts with TRXz to regulate chloroplast development in rice

Chloroplast genes are transcribed by the plastidencoded RNA polymerase（PEP） or nucleus-encoded RNA polymerase. FRUCTOKINASE-LIKE PROTEINS（FLNs） are phosphofructokinase-B（Pfk B）-type carbohydrate kinases that act as part of the PEP complex; however, the molecular mechanisms underlying FLN activity in rice remain elusive.Previously, we identified and characterized a heat-stress sensitive albino（hsa_1） mutant in rice. Map-based cloning revealed that HSA_1 encodes a putative OsFLN_2. Here, we further demonstrated that knockdown or knockout of the OsFLN_1, a close homolog of HSA_1/OsFLN_2, considerably inhibits chloroplast biogenesis and the fln_1 knockout mutants, created by clustered regularly interspaced short palindromic repeats（CRISPR） and CRISPR-associate protein_9, exhibit severe albino phenotype and seedling lethality. Moreover, OsFLN_1 localizes to the chloroplast.Yeast two-hybrid, pull-down and bimolecular fluorescencecomplementation experiments revealed that OsFLN_1 and HSA_1/OsFLN_2 interact with THIOREDOXINZ（OsTRXz） to regulate chloroplast development. In agreement with this,knockout of OsTRXz resulted in a similar albino and seedling lethality phenotype to that of the fln_1 mutants. Quantitative reverse transcription polymerase chain reaction and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in fln_1 and trxz mutants, indicating that loss of OsFLN_1, HSA_1/OsFLN_2, or OsTRXz function perturbs the stability of the transcriptionally active chromosome complex and PEP activity. These results show that OsFLN_1 and HSA_1/OsFLN_2 contribute to chloroplast biogenesis and plant growth.