Exploring the regulatory role of non-coding RNAs in fiber development and direct regulation of GhKCR2 in the fatty acid metabolic pathway in upland cotton.

Cotton fiber holds immense importance as the primary raw material for the textile industry. Consequently, comprehending the regulatory mechanisms governing fiber development is pivotal for enhancing fiber quality. Our study aimed to construct a regulatory network of competing endogenous RNAs (ceRNAs) and assess the impact of non-coding RNAs on gene expression throughout fiber development. Through whole transcriptome data analysis, we identified differentially expressed genes (DEGs) regulated by non-coding RNA (ncRNA) that were predominantly enriched in phenylpropanoid biosynthesis and the fatty acid elongation pathway. This analysis involved two contrasting phenotypic materials (J02-508 and ZRI015) at five stages of fiber development. Additionally, we conducted a detailed analysis of genes involved in fatty acid elongation, including KCS, KCR, HACD, ECR, and ACOT, to unveil the factors contributing to the variation in fatty acid elongation between J02-508 and ZRI015. Through the integration of histochemical GUS staining, dual luciferase assay experiments, and correlation analysis of expression levels during fiber development stages for lncRNA MSTRG.44818.23 (MST23) and GhKCR2, we elucidated that MST23 positively regulates GhKCR2 expression in the fatty acid elongation pathway. This identification provides valuable insights into the molecular mechanisms underlying fiber development, emphasizing the intricate interplay between non-coding RNAs and protein-coding genes.

Identification and characterization of candidate genes for primary root length in Asiatic cotton (Gossypium arboreum L.).

KEY MESSAGE: One major gene controlling primary root length (PRL) in Gossypium arboreum is identified and this research provides a theoretical basis for root development for cotton. Primary root elongation is an essential process in plant root system structure. Here, we investigated the primary root length (PRL) of 215 diploid cotton (G. arboreum) accessions at 5, 8, 10, 15 days after sowing. A Genome-wide association study was performed for the PRL, resulting in 49 significant SNPs associated with 32 putative candidate genes. The SNP with the strongest signal (Chr07_8047530) could clearly distinguish the PRLs between accessions with two haplotypes. GamurG is the only gene that showed higher relative expression in the long PRL genotypes than the short PRL genotypes, which indicated it was the most likely candidate gene for regulating PRL. Moreover, the GamurG-silenced cotton seedlings showed a shorter PRL, while the GamurG-overexpressed Arabidopsis exhibited a significantly longer PRL. Our findings provide insight into the regulation mechanism of cotton root growth and will facilitate future breeding programs to optimize the root system structure in cotton.

Injectable Bioadhesive Photocrosslinkable Hydrogels with Sustained Release of Kartogenin to Promote Chondrogenic Differentiation and Partial-Thickness Cartilage Defects Repair.

Partial-thickness cartilage defect (PTCD) is a common and formidable clinical challenge without effective therapeutic approaches. The inherent anti-adhesive characteristics of the extracellular matrix within cartilage pose a significant impediment to the integration of cells or biomaterials with the native cartilage during cartilage repair. Here, an injectable photocrosslinked bioadhesive hydrogel, consisting of gelatin methacryloyl (GM), acryloyl-6-aminocaproic acid-g-N-hydroxysuccinimide (AN), and poly(lactic-co-glycolic acid) microspheres loaded with kartogenin (KGN) (abbreviated as GM/AN/KGN hydrogel), is designed to enhance interfacial integration and repair of PTCD. After injected in situ at the irregular defect, a stable and robust hydrogel network is rapidly formed by ultraviolet irradiation, and it can be quickly and tightly adhered to native cartilage through amide bonds. The hydrogel exhibits good adhesion strength up to 27.25 ± 1.22 kPa by lap shear strength experiments. The GM/AN/KGN hydrogel demonstrates good adhesion, low swelling, resistance to fatigue, biocompatibility, and chondrogenesis properties in vitro. A rat model with PTCD exhibits restoration of a smoother surface, stable seamless integration, and abundant aggrecan and type II collagen production. The injectable stable adhesive hydrogel with long-term chondrogenic differentiation capacity shows great potential to facilitate repair of PTCD.

Genomic loci associated with leaf abscission contribute to machine picking and environmental adaptability in upland cotton (Gossypium hirsutum L.).

INTRODUCTION: Defoliation by applying defoliants before machine picking is an important agricultural practice that enhances harvesting efficiency and leads to increased raw cotton purity. However, the fundamental characteristics of leaf abscission and the underlying genetic basis in cotton are not clearly understood. OBJECTIVES: In this study, we aimed to (1) reveal the phenotypic variations in cotton leaf abscission, (2) discover the whole-genome differentiation sweeps and genetic loci related to defoliation, (3) identify and verify the functions of key candidate genes associated with defoliation, and (4) explore the relationship between haplotype frequency of loci and environmental adaptability. METHODS: Four defoliation-related traits of 383 re-sequenced Gossypium hirsutum accessions were investigated in four environments. The genome-wide association study (GWAS), linkage disequilibrium (LD) interval genotyping and functional identification were conducted. Finally, the haplotype variation related to environmental adaptability and defoliation traits was revealed. RESULTS: Our findings revealed the fundamental phenotypic variations of defoliation traits in cotton. We showed that defoliant significantly increased the defoliation rate without incurring yield and fiber quality penalties. The strong correlations between defoliation traits and growth period traits were observed. A genome-wide association study of defoliation traits identified 174 significant SNPs. Two loci (RDR7 on A02 and RDR13 on A13) that significantly associated with the relative defoliation rate were described, and key candidate genes GhLRR and GhCYCD3;1, encoding a leucine-rich repeat (LRR) family protein and D3-type cell cyclin 1 protein respectively, were functional verified by expression pattern analysis and gene silencing. We found that combining of two favorable haplotypes (HapRDR7 and HapRDR13) improved sensitivity to defoliant. The favorable haplotype frequency generally increased in high latitudes in China, enabling adaptation to the local environment. CONCLUSION: Our findings lay an important foundation for the potentially broad application of leveraging key genetic loci in breeding machine-pickable cotton.

Epigenetic insight into floral transition and seed development in plants.

Seasonal changes are crucial in shifting the developmental stages from the vegetative phase to the reproductive phase in plants, enabling them to flower under optimal conditions. Plants grown at different latitudes sense and interpret these seasonal variations, such as changes in day length (photoperiod) and exposure to cold winter temperatures (vernalization). These environmental factors influence the expression of various genes related to flowering. Plants have evolved to stimulate a rapid response to environmental conditions through genetic and epigenetic mechanisms. Multiple epigenetic regulation systems have emerged in plants to interpret environmental signals. During the transition to the flowering phase, changes in gene expression are facilitated by chromatin remodeling and small RNAs interference, particularly in annual and perennial plants. Key flowering regulators, such as FLOWERING LOCUS C (FLC) and FLOWERING LOCUS T (FT), interact with various factors and undergo chromatin remodeling in response to seasonal cues. The Polycomb silencing complex (PRC) controls the expression of flowering-related genes in photoperiodic flowering regulation. Under vernalization-dependent flowering, FLC acts as a potent flowering suppressor by downregulating the gene expression of various flower-promoting genes. Eventually, PRCs are critically involved in the regulation of FLC and FT locus interacting with several key genes in photoperiod and vernalization. Subsequently, PRCs also regulate Epigenetical events during gametogenesis and seed development as a driving force. Furthermore, DNA methylation in the context of CHG, CG, and CHH methylation plays a critical role in embryogenesis. DNA glycosylase DME (DEMETER) is responsible for demethylation during seed development. Thus, the review briefly discusses flowering regulation through light signaling, day length variation, temperature variation and seed development in plants.

Physiological and transcriptional analyses reveal formation of memory under recurring drought stresses in seedlings of cotton (Gossypium hirsutum).

Plants are frequently subjected to a range of environmental stresses, including drought, salinity, cold, pathogens, and herbivore attacks. To survive in such conditions, plants have evolved a novel adaptive mechanism known as 'stress memory'. The formation of stress memories necessitates coordinated responses at the cellular, genetic/genomic, and epigenetic levels, involving altered physiological responses, gene activation, hyper-induction and chromatin modification. Cotton (Gossypium spp.) is an important economic crop with numerous applications and high economic value. In this study, we establish G. hirsutum drought memory following cycles of mild drought and re-watering treatments and analyzed memory gene expression patterns. Our findings reveal the physiological, biochemical, and molecular mechanisms underlying drought stress memory formation in G. hirsutum. Specifically, H3K4me3, a histone modification, plays a crucial role in regulating [+ /+ ] transcriptional memory. Moreover, we investigated the intergenerational inheritance of drought stress memory in G. hirsutum. Collectively, our data provides theoretical guidance for cotton breeding.

Transcriptomic analysis reveals the beneficial effects of salt priming on enhancing defense responses in upland cotton under successive salt stress.

Priming-mediated stress tolerance in plants stimulates defense mechanisms and enables plants to cope with future stresses. Seed priming has been proven effective for tolerance against abiotic stresses; however, underlying genetic mechanisms are still unknown. We aimed to assess upland cotton genotypes and their transcriptional behaviors under salt priming and successive induced salt stress. We pre-selected 16 genotypes based on previous studies and performed morpho-physiological characterization, from which we selected three genotypes, representing different tolerance levels, for transcriptomic analysis. We subjected these genotypes to four different treatments: salt priming (P0), salt priming with salinity dose at 3-true-leaf stage (PD), salinity dose at 3-true-leaf stage without salt priming (0D), and control (CK). Although the three genotypes displayed distinct expression patterns, we identified common differentially expressed genes (DEGs) under PD enriched in pathways related to transferase activity, terpene synthase activity, lipid biosynthesis, and regulation of acquired resistance, indicating the beneficial role of salt priming in enhancing salt stress resistance. Moreover, the number of unique DEGs associated with G. hirsutum purpurascens was significantly higher compared to other genotypes. Coexpression network analysis identified 16 hub genes involved in cell wall biogenesis, glucan metabolic processes, and ribosomal RNA binding. Functional characterization of XTH6 (XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE) using virus-induced gene silencing revealed that suppressing its expression improves plant growth under salt stress. Overall, findings provide insights into the regulation of candidate genes in response to salt stress and the beneficial effects of salt priming on enhancing defense responses in upland cotton.

Multimodal steerable earthworm-inspired soft robot based on vacuum and positive pressure powered pneumatic actuators.

This article presents a multimodal steerable earthworm-inspired soft robot based on vacuum and positive pressure powered pneumatic actuators capable of crawling both inside pipelines and on planar surfaces. The optimized modular vacuum pressure-driven actuator can generate deformation and anchoring motion through a unified structure under low vacuum pressure, giving it significant speed advantages and multi-modal locomotion capabilities. Meanwhile, the positive pressure powered actuator (PPPA) enables the robot to achieve controlled multi-directional and multi-degrees-of-freedom steering, moreover, enhances the consistency of the driving mechanism. The incorporation of front-end pressure sensing enables the robot to autonomously detect and evaluate pressure, facilitating automatic obstacle avoidance through the activation of corresponding turning units of PPPA. In the process of optimizing motion parameters, the overall motion efficiency has been improved by 16.7% by improving the control law. Through adjustments and optimizations of the interval time (cycle time), the robot is able to achieve a speed of 7.16 mm s-1during planar locomotion and 1.94 mm s-1during in-pipe locomotion. Using the developed robot, we conducted a series of turning experiments, including surface obstacle avoidance and cross-plane crawling, which demonstrated its enhanced capability in cross-plane steering and locomotion. Its related speed indicators showcase superior overall performance compared to other developed robots of the same type.

Incidence, Predictors, and Strategies for Retrograde Wire Tracking Failure via Poor Septal Collateral Channels in Chronic Total Occlusion.

Background: Retrograde guidewire (GW) tracking success via a poor septal collateral channel (CC) when an antegrade approach fails is crucial for successful revascularization of coronary chronic total occlusion (CTO) with poor septal CC. However, the incidence, predictors, and management strategies for retrograde GW tracking failure via poor septal CC remain unclear. Methods: In total, 122 CTO patients who underwent retrograde septal percutaneous coronary intervention (PCI) with poor CC between January 2017 and May 2022 were retrospectively analyzed. Patients were divided into the retrograde GW tracking success group (success group) and the retrograde GW tracking failure group (failure group). Clinical and angiographic data were compared to investigate the predictors of retrograde GW tracking failure. Results: The incidence of GW tracking failure was 22.1% (27/122). Patients in the failure group had a higher prevalence of left anterior descending artery (LAD) CTO (66.7% vs 37.9%; p = 0.009) and a higher incidence of well-developed non-septal collateral (66.7% vs 30.5%; p = 0.001). Patients with a septal CC diameter ≥ 1 mm (48.1% vs 70.5%; p = 0.040), ≥ 3 septal CCs (44.4% vs 66.3%; p = 0.046), and initial retrograde application of Guidezilla (37.0% vs 60.0%; p = 0.048) were significantly lower in the failure group than in the success group. The binary logistics regression model showed that a CC diameter < 1 mm, well-developed non-septal collateral, and LAD CTO were independent predictors for GW tracking failure in patients undergoing retrograde CTO PCI via poor septal CC. Conclusion: The success rate of retrograde GW tracking via poor septal CC was high, with a relatively high procedural success rate. A CC diameter < 1 mm, well-developed non-septal collateral, and LAD CTO were independent predictors of GW tracking failure in patients undergoing retrograde CTO PCI via poor septal CC.

Preoperative small pulmonary nodule localisation using hookwires or coils: strategy selection in adverse events.

OBJECTIVE: This is a retrospective study of adverse events associated with preoperative computed tomography (CT)-guided hookwire or coil localisation. We analysed the experience and process flaws in resecting ground-glass nodules (GGNs) through video-assisted thoracoscopic surgery (VATS) and determined the remedial strategy. METHODS: Adverse events were evaluated in 40 patients with 45 GGNs who underwent CT-guided hookwire or coil localisation before VATS. For lesions not successfully marked or detected, palpation, resection of the highly suspected area, segmentectomy or lobectomy was performed. RESULTS: Among all adverse events, 15 were dislodgement of the marking materials, 5 were breakaway of the marking materials, 7 were > 2 cm distance between the lesions and the tips, 3 was marking material across the two adjacent lobes, 15 were pneumothorax and 2 were certain parts of marking materials stuck into the chest wall. All GGNs were resected successfully. 20 lesions were detected by palpation. 9 GGNs were discovered after the resection of highly suspected areas. Segmentectomies and lobectomies were performed directly on 7 and 9 GGNs, respectively. CONCLUSIONS: When adverse events occur, a second intraoperative localisation, by resecting the highly suspected area either through non-anatomical resection (wedge resection) or anatomical resection (segmentectomy or lobectomy) using the VATS should be considered the alternatives for GGNs localization.

Crystal Growth, Characterization, and Properties of Nonlinear Optical Crystals of LixAg1-xGaSe2 for Mid-Infrared Applications.

LixAg1-xGaSe2 is a new series of solid solution crystals that has a large nonlinear optical (NLO) coefficient and laser-induced damage threshold (LIDT). It has great application prospects in mid-infrared laser frequency conversion. In this work, LixAg1-xGaSe2 (x = 0, 0.2, 0.4, 0.5, 0.6, 0.8, and 1) crystals (Φ 16 mm × 40 mm) were grown by the improved Bridgeman method in a four-zone furnace. It is found that the LixAg1-xGaSe2 (x = 0.2-0.8) crystals keep the same tetragonal symmetry with AgGaSe2 and the melting and solidification temperature increase with the Li content. Because the as-grown crystals are almost opaque in the visible-NIR range, an annealing experiment is necessary. After annealing, the transmittance is improved significantly, which can meet the application requirements. The band gap is changed by annealing atmosphere; for instance, the band gap of Li0.6Ag0.4GaSe2 annealed in a LiGaSe2 powder atmosphere increases from 2.35 to 2.56 eV, while the band gap of LiGaSe2 annealed in vacuum decreases from 3.39 to 3.01 eV. Finally, the LixAg1-xGaSe2 shows an extreme SHG response, especially Li0.8Ag0.2GaSe2, which has about five times that of LiGaSe2, proving the promising NLO properties.

Genome-wide analysis of the serine carboxypeptidase-like protein family reveals Ga09G1039 is involved in fiber elongation in cotton.

The Gossypium is a model genus for understanding polyploidy and the evolutionary pattern of inheritance. This study aimed to investigate the characteristics of SCPLs in different cotton species and their role in fiber development. A total of 891 genes from one typical monocot and ten dicot species were naturally divided into three classes based on phylogenetic analysis. The SCPL gene family in cotton has undergone intense purifying selection with some functional variation. Segmental duplication and whole genome duplication were shown to be the two main reasons for the increase in the number of genes during cotton evolution. The identification of Gh_SCPL genes exhibiting differential expression in particular tissues or response to environmental stimuli provides a new measure for the in-depth characterization of selected genes of importance. Ga09G1039 was involved in the developmental process of fibers and ovules, and it is significantly different from proteins from other cotton species in terms of phylogenetic, gene structure, conserved protein motifs and tertiary structure. Overexpression of Ga09G1039 significantly increased the length of stem trichomes. Ga09G1039 may be a serine carboxypeptidase protein with hydrolase activity, according to functional region, prokaryotic expression, and western blotting analysis. The results provide a comprehensive overview of the genetic basis of SCPLs in Gossypium and further our knowledge in understanding the key aspects of SCPLs in cotton with their potential role in fiber development and stress resistance.

Engineering the Staple Oil Crop Brassica napus Enriched with α-Linolenic Acid Using the Perilla FAD2-FAD3 Fusion Gene.

Modern people generally suffer from α-linolenic acid (ALA) deficiency, since most staple food oils are low in ALA content. Thus, the enhancement of ALA in staple oil crops is of importance. In this study, the FAD2 and FAD3 coding regions from the ALA-king species Perilla frutescens were fused using a newly designed double linker LP4-2A, driven by a seed-specific promoter PNAP, and engineered into a rapeseed elite cultivar ZS10 with canola quality background. The mean ALA content in the seed oil of PNAP:PfFAD2-PfFAD3 (N23) T5 lines was 3.34-fold that of the control (32.08 vs 9.59%), with the best line being up to 37.47%. There are no significant side effects of the engineered constructs on the background traits including oil content. In fatty acid biosynthesis pathways, the expression levels of structural genes as well as regulatory genes were significantly upregulated in N23 lines. On the other hand, the expression levels of genes encoding the positive regulators of flavonoid-proanthocyanidin biosynthesis but negative regulators of oil accumulation were significantly downregulated. Surprisingly, the ALA level in PfFAD2-PfFAD3 transgenic rapeseed lines driven by the constitutive promoter PD35S was not increased or even showed a slight decrease due to the lower level of foreign gene expression and downregulation of the endogenous orthologous genes BnFAD2 and BnFAD3.

Crystal Growth, Thermal Treatment, and Characterization of Nonlinear Optical Crystal LiGa0.5In0.5Se2 for Mid-infrared Applications.

LiGa0.5In0.5Se2 is a new quaternary nonlinear optical crystal for the mid-IR application grown as a mixed crystal of the LiGaSe2-LiInSe2 solid-solution system. It is transparent in the 0.47-14 µm range and has an appropriate bandgap and a lower melting point than LiGaSe2 and LiInSe2. It is more technological about the growth process since its homogeneity range is broader in the phase diagram. In this work, we have synthesized the LiGa0.5In0.5Se2 polycrystal by the two-zone temperature method. LiGa0.5In0.5Se2 single crystals (Φ26 mm × 50 mm) were grown through the modified Bridgman method with the c-axis seed crystal which has the smallest thermal expansion coefficient of the three main axes in 293-773 K. The crystal structure was studied by X-ray diffraction and the Rietveld refinement method. Due to the low transmittance of the as-grown crystals, a systematic thermal treatment experiment was carried out. In the annealing experiment, the crystal surface is seriously enriched with selenium due to the thermal diffusion of selenium, resulting in the crystal opacity and cracking, while after vacuum quenching at 873 K, the transmittance of the LiGa0.5In0.5Se2 crystal wafer was greatly improved, the bandgap shows a large increase from 2.13 to 2.51 eV, and the quenched crystal shows strong SHG response (×1.91 LiGaSe2). The chemical states and vibration modes of surface elements for both conditions were characterized by X-ray photoelectron and Raman spectra. Density functional theory calculations were carried out to simulate the phonon spectrum and phonon density of states, which can help to study the phonon vibration modes in the lattice.

Remote preconditioning combined with nebulized budesonide alleviate lipopolysaccharide induced acute lung injury via regulating HO-1 and NF-κB in rats.

BACKGROUND: Acute lung injury (ALI) may result in severe systemic inflammation and is life-threatening. Remote inflammatory preconditioning (RIPC) has been confirmed to have an endogenous protective effect against ALI. Budesonide (BS) is a potent corticosteroid typically administered through nebulization that reduces inflammation in the lungs. We speculate that the combined use of RIPC and nebulized BS has a stronger protective effect on ALI. METHODS: 48 Sprague-Dawley male rats were used for the experiments. Animals were divided evenly and randomly into three groups, control (NS injection), LPS (LPS injection), and RIPC (LPS injection with RIPC). Each group was then divided into two subgroups with inhalation of nebulized normal saline (NS) or BS. Prior to injection of LPS, RIPC was performed by tying and untying the right hind limb for three cycles of 5 min each. Following LPS injection, animals in each subgroup were placed in a same cage for nebulized inhalation. Animals were sacrificed 6 h after LPS injection. Histological evaluation of ALI and lung wet-to-dry weight ratio were measured. Serum lactate acid, inflammatory cytokines, oxidative stress indicators were detected. The expression of HO-1, NF-κB p65 and p-p65 was measured by western blotting. RESULTS: RIPC combined with nebulized BS significantly attenuated the LPS-induced ALI in rats. Reduction of MDA, increasing of SOD activity were found significantly improved by the joint strategy. TNF- and IL-1ß rise brought on by LPS was reduced, but IL-10 production dramatically enhanced when compared to the LPS group. The expression of HO-1 was significantly increased by RIPC combined with nebulized BS while the expression of NF-κB p65 and p-p65 was decreased when compared with the LPS group. CONCLUSION: RIPC combined with nebulized budesonide is protective for ALI induced by LPS in rats.

Silencing of GhORP_A02 enhances drought tolerance in Gossypium hirsutum.

BACKGROUND: ORP (Oxysterol-binding protein-related proteins) genes play a role in lipid metabolism, vesicular transferring and signaling, and non-vesicular sterol transport. However, no systematic identification and analysis of ORP genes have been reported in cotton. RESULT: In this study, we identified 14, 14, 7, and 7 ORP genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Phylogenetic analysis showed that all ORP genes could be classified into four groups. Gene structure and conserved motif analysis suggest that the function of this gene family was conserved. The Ka/Ks analysis showed that this gene family was exposed to purifying selection during evolution. Transcriptome data showed that four ORP genes, especially GhORP_A02, were induced by abiotic stress treatment. The cis-acting elements in the ORP promoters were responsive to phytohormones and various abiotic stresses. The silenced plants of GhORP_A02 were more sensitive to drought stress when compared to control. CONCLUSION: The major finding of this study shed light on the potential role of ORP genes in abiotic stress and provided a fundamental resource for further analysis in cotton.

Bioprinted living tissue constructs with layer-specific, growth factor-loaded microspheres for improved enthesis healing of a rotator cuff.

Substantial challenges remain in constructing the native tendon-to-bone interface for rotator cuff healing owing to the enthesis tissues' highly organized structural and compositional gradients. Herein, we propose to bioprint living tissue constructs with layer-specific growth factors (GFs) to promote enthesis regeneration by guiding the zonal differentiation of the loaded stem cells in situ. The sustained release of tenogenic, chondrogenic, and osteogenic GFs was achieved via microsphere-based delivery carriers embedded in the bioprinted constructs. Compared to the basal construct without GFs, the layer-specific tissue analogs realized region-specific differentiation of stem cells in vitro. More importantly, bioprinted living tissue constructs with layer-specific GFs rapidly enhanced the enthesis regeneration in a rabbit rotator cuff tear model in terms of biomechanical restoration, collagen deposition, and alignment, showing gradient interface of fibrocartilage structures with aligned collagen fibrils and an ultimate load failure of 154.3 ± 9.5 N resembling those of native enthesis tissues in 12 weeks. This exploration provides a feasible strategy to engineer living tissue constructions with region-specific differentiation potentials for the functional repair of gradient enthesis tissues. STATEMENT OF SIGNIFICANCE: Previous studies that employed acellular layer-specific scaffolds or stem cells for the reconstruction of the rotator cuff faced challenges due to their insufficient capability to rebuild the anisotropic compositional and structural gradients of native enthesis tissues. This manuscript proposed a living tissue construct with layer-specific, GFs-loaded µS, which can direct in situ and region-specific differentiation of the embedded stem cells to tenogenic, chondrogenic, and osteogenic lineages for functional regeneration of the enthesis tissues. This bioprinted living tissue construct with the unique capability to reduce fibrovascular scar tissue formation and simultaneously facilitate enthesis tissue remodeling might provide a promising strategy to repair complex and gradient tissues in the future.