Yang Huang

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in several biological processes, including the regulation of body color. Leopard coral grouper (Plectropomus leopardus) is a valuable marine aquaculture fish; however, at present, there are no published reports on its early body color development. The skin color of P. leopardus undergoes a transition from transparent to red from 26 days post-hatching (dph) to 30 dph. In this study, we performed miRNA sequencing on 26 dph (Transparent, PT) and 30 dph (Red, PR) skin samples of P. leopardus to explore the molecular mechanism underlying red color formation. A total of 44.31 M and 37.55 M clean tags were obtained from PT and PR group, respectively. Among these tags, 981 miRNAs were identified, including 493 known and 488 novel miRNAs. A total of 106 differentially expressed miRNAs (DEMs) were identified in PT vs. PR, with 43 up-regulated and 63 down-regulated miRNAs in the PR group, compared to the PT group (|fold change| > 2 and p-value < 0.05). A miRNA-mRNA network based on 18 candidate miRNAs and 53 target genes related to pigmentation, and KEGG enrichment analysis of the target genes of all DEMs, revealed that miRNAs involved in the formation of red skin color were mainly related to: 1) the inhibition of melanin synthesis (miR-141-z, miR-206-z, miR-206-y, miR-27-z, miR-137-y, miR-204-x, miR-204-y, miR-211-x, miR-211-z); 2) chromatophore development (miR-206-z, miR-206-y, miR-499-y, miR-1-z, miR-2188-x, miR-423-x); and 3) carotenoid metabolism (miR-204-x, miR-204-y, miR-499-y). This study demonstrates the potential role of miRNAs in red color formation and lays the foundation for the molecular mechanism of body color polymorphism in P. leopardus.

Anterior cruciate ligament (ACL) rupture is one of the most common knee injuries. Due to the limited self-healing capacity, complex structure, and mechanical environment, the regeneration of ACL remains a challenge. To achieve fast recovery and long-term ACL regeneration, biodegradable scaffolds with excellent initial mechanical support and cell recruitment ability have gained increasing research interest. The aptamer-functionalized silk fiber was fabricated by embedding silk fiber into morpholinoethanesulfonic/1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride, N-hydroxysuccinimide buffer with aptamer 19s (Apt19s) and characterized in vitro. The clinically size-matched silk ligament was braided utilizing commercial methodology (polyethylene terephthalate ligament), and the tensile strength was analyzed. A total of 18 rabbits were used to test the early mesenchymal stem cell (MSC) recruitment of Apt19s-functionalized silk ligaments. Macroscopic analysis and hematoxylin and eosin and immunohistochemistry stainings were applied at 3, 5, and 7 days post-implantation. The ultimate strength of silk fiber was lower than that of PET fibers. However, the prepared silk ligament had comparable mechanical strength to the native ACL. In vitro, the Apt19s-functionalized fiber recruited bone marrow-derived MSCs. In vivo, Apt19s-functionalized silk ligament enhanced early MSCs recruitment after implantation in comparison with the aptamer-free ligament. The Apt19s-functionalized scaffold silk ligament exhibited comparable mechanical strength with native ACL and enhanced endogenous MSC homing.

To establish a method for investigating the permeability of calcified cartilage zone (CCZ) and to observe solute transport between articular cartilage (AC) and subchondral bone (SB) through intact CCZ in vivo. We developed a novel fixing device combined with un-decalcified fluorescence observation method to address the permeability of CCZ in live mice. Twenty-four Balb/c female mice aged 1 to 8 months were used to observe the development of CCZ. Eighty-four Balb/c female mice (aged 1 or 6 months) with mature or immature CCZ of distal femur were used to investigate the permeability of intact CCZ in vivo. Diffusivity of rhodamine B (476 Da) and tetramethyl-rhodamine isothicyanate-dextran (TRITC-Dextran, 20 kDa) was tested from AC to SB in 0 minutes, 1 minute, 15 minutes, 30 minutes, 1 hour, and 2 hours. None diffused knee joints (0 minutes) served as blank control, while in vitro immersion of distal femurs in rhodamine B or TRITC-Dextran for 72 hours served as positive control. CCZ was well developed in 6-month mice. Both tracers penetrated immature CCZ down to SB in less than 1 hour in live mice, while the diffusion of both tracers decreased rapidly at tidemark in all testing time points. Current study provided direct evidence of blocking effect of CCZ in solute transportation during short diffusion period in live animal, indicating the important role of CCZ in joint development and microenvironment maintenance.

Ca²⁺ has been recognized as a key molecule for chondrocytes, however, the role and mechanism of spontaneous [Ca ²⁺] _i signaling in cartilaginous extracellular matrix (ECM) metabolism regulation are unclear. Here we found that spontaneous Ca ²⁺ signal of in‐situ porcine chondrocytes was [Ca ²⁺] _o dependent, and mediated by [Ca ²⁺] _i store release. T‐type voltage‐dependent calcium channel (T‐VDCC) mediated [Ca ²⁺] _o influx was associated with decreased cell viability and expression levels of ECM deposition genes. Further analysis revealed that chondrocytes expressed both inositol 1,4,5‐trisphosphate receptor (InsP3R) and Orai isoforms. Inhibition of endoplasmic reticulum (ER) Ca ²⁺ release and store‐operated calcium entry significantly abolished spontaneous [Ca ²⁺] _i signaling of in‐situ chondrocytes. Moreover, blocking ER Ca ²⁺ release with InsP3R inhibitors significantly upregulated ECM degradation enzymes production, and was accompanied by decreased proteoglycan and collagen type II intensity. Taken together, our data provided evidence that spontaneous [Ca ²⁺] _i signaling of in‐situ porcine chondrocytes was tightly regulated by [Ca ²⁺] _o influx, InsP3Rs mediated [Ca ²⁺] _i store release, and Orais mediated calcium release‐activated calcium channels activation. Both T‐VDCC mediated [Ca ²⁺] _o influx and InsP3Rs mediated ER Ca ²⁺ release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.

Osteoarthritis (OA) is the most prevalent form of joint disease and lacks effective treatment. Cell-based therapy through intra-articular injection holds great potential for effective intervention at its early stage. Despite the promising outcomes, major barriers for successful clinical application such as lack of specific targeting of transplanted cells still remain. Here, novel polyethylenimine-wrapped iron oxide nanoparticles (PEI/IONs) were utilized as a magnetic agent, and the in vitro efficiency of PEI/ION labeling, and the influence on the chondrogenic properties of chondrocytes were evaluated; the in vivo feasibility of magnetic-targeting intra-articular injection with PEI/ION labeled autologous chondrocytes was investigated using a rabbit articular cartilage defect model. Our data showed that chondrocytes were conveniently labeled with PEI/IONs in a time- and dose-dependent manner, while the viability was unaffected. No significant decrease in collagen type-II synthesis of labeled chondrocytes was observed at low concentration. Macrographic and histology evaluation at 1 week post intra-articular injection revealed efficient cell delivery at chondral defect sites in the magnetic-targeting group. In addition, chondrocytes in the defect area presented a normal morphology, and the origin of cells within was confirmed by immunohistochemistry staining against BrdU and Prussian blue staining. The present study shows proof of concept experiments in magnetic-targeting of PEI/ION labeled chondrocytes for articular cartilage repair, which might provide new insight to improve current cartilage repair strategies.