[A dual-crosslinked injectable hydrogel derived from muscular decellularized matrix promoting myoblasts proliferation and myogenic differentiation].

Objective: To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. Methods: Firstly, hyaluronic acid was oxidized with NaIO 4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). Results: FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P<0.05). Conclusion: The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.

[Research of diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity for promoting in vivo cartilage regeneration].

Objective: To develop a diclofenac sodium-loaded gelatin scaffold with anti-inflammatory activity and provide a new avenue for alleviating the inflammatory response and enhancing cartilage regeneration in vivo. Methods: Diclofenac sodium was homogeneously mixed with gelatin to prepare a diclofenac sodium-loaded porous gelatin scaffold by freeze-drying method as the experimental group, and a pristine porous gelatin scaffold was served as a control group. The general morphology of the scaffold was observed, the pore size of the scaffold was measured by scanning electron microscopy, the porosity of the scaffold was calculated by drainage method, the loading of diclofenac sodium into the gelatin scaffold was detected by fourier transform infrared spectrometer and X-ray diffraction examinations, and the release kinetics of diclofenac sodium from gelatin scaffold was tested using an in vitro release assay. The two scaffolds were co-cultured with lipopolysaccharide-predisposed RAW264.7 in vitro, and the expressions of interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immuno sorbent assay, and Western blot, to detect the in vitro anti-inflammatory effect of the drug-loaded scaffold. Thereafter, the second generation chondrocytes of New Zealand white rabbits were inoculated on the two groups of scaffolds for in vitro culture, and the cytocompatibility of the scaffold was tested by live/dead staining and cell counting kit 8 assay, the feasibility of in vitro cartilage regeneration of the scaffold was evaluated via gross observation, HE staining, Safranin-O staining, and immunohistochemical collagen type Ⅱ staining, as well as biochemical quantitative analyses. Finally, the two groups of chondrocyte-scaffolds were implanted subcutaneously into New Zealand white rabbits, and after 4 weeks, the general observation, HE staining, safranin O staining, immunohistochemical collagen type Ⅱ staining, and biochemical quantitative analyses were performed to verify the cartilage regeneration in vivo, and the expression of inflammation-related genes CD3 and CD68 was detected by RT-PCR to comprehensively evaluate the anti-inflammatory performance of the scaffolds in vivo. Results: The two scaffolds exhibited similar gross, microporous structure, pore size, and porosity, showing no significant difference (P>0.05). Diclofenac sodium was successfully loaded into gelatin scaffold. Data from in vitro anti-inflammatory assay suggested that diclofenac sodium-loaded gelatin scaffold showed alleviated gene and protein expressions of IL-1ß and TNF-α when compared with gelatin scaffold (P<0.05). The evaluation of cartilage regeneration in vitro showed that the number of living cells increased significantly with the extension of culture time, and there was no significant difference between the two groups at each time point (P>0.05). White cartilage-like tissue was regenerated from the scaffolds in both groups, histological observation showed typical cartilage lacuna structure and specific cartilage extracellular matrix secretion. There was no significant difference in the content of cartilage-specific glycosaminoglycan (GAG) and collagen type Ⅱ between the two groups (P>0.05). In vivo experiments showed that the samples in the experimental group had porcelain white cartilage like morphology, histologic staining showed obvious cartilage lacuna structure and cartilage specific extracellular matrix, the contents of GAG and collagen type Ⅱ were significantly higher than those in the control group, and the protein and mRNA expressions of CD3 and CD68 were significantly lower than those in the control group, with significant differences (P<0.05). Conclusion: The diclofenac sodium-loaded gelatin scaffold presents suitable pore size, porosity, and cytocompatibility, as well as exhibited satisfactory anti-inflammatory ability, providing a reliable scheme for alleviating the inflammatory reaction of regenerated cartilage tissue after in vivo implantation and promoting cartilage regeneration in vivo.

Icariin-loaded hydrogel with concurrent chondrogenesis and anti-inflammatory properties for promoting cartilage regeneration in a large animal model.

Currently, an effective repair method that can promote satisfactory cartilage regeneration is unavailable for cartilage damages owing to inevitable inflammatory erosion. Cartilage tissue engineering has revealed considerable treatment options for cartilage damages. Icariin (ICA) is a flavonoid component of Epimedii folium with both chondrogenic and anti-inflammatory properties. In this study, we prepared an ICA/CTS hydrogel by loading ICA into chitosan (CTS) hydrogel to impart chondrogenesis and anti-inflammatory properties to the ICA/CTS hydrogel. In vitro results revealed that ICA showed sustained release kinetics from the ICA/CTS hydrogel. In addition, compared to the CTS hydrogel, the ICA/CTS hydrogel exhibited a favorable in vitro anti-inflammatory effect upon incubation with lipopolysaccharide pre-induced RAW264.7 macrophages, as indicated by the suppression of inflammatory-related cytokines (IL-6 and TNF-α). Additionally, when co-cultured with chondrocytes in vitro, the ICA/CTS hydrogel showed good cytocompatibility, accelerated chondrocyte proliferation, and enhanced chondrogenesis compared to the CTS hydrogel. Moreover, the in vitro engineered cartilage from the chondrocyte-loaded ICA/CTS hydrogel achieved stable cartilage regeneration when subcutaneously implanted in a goat model. Finally, the addition of ICA endowed the ICA/CTS hydrogel with a potent anti-inflammatory effect compared to what was observed in the CTS hydrogel, as confirmed by the attenuated IL-1ß, IL-6, TNF-α, and TUNEL expression. The prepared ICA/CTS hydrogel offered an effective method of delivery for chondrogenic and anti-inflammatory agents and served as a useful platform for cartilage regeneration in an immunocompetent large animal model.

[Effect of xanthohumol-loaded anti-inflammatory scaffolds on cartilage regeneration in goats].

Objective: To develop an anti-inflammatory poly (lactic-co-glycolic acid) (PLGA) scaffold by loading xanthohumol, and investigate its anti-inflammatory and cartilage regeneration effects in goats. Methods: The PLGA porous scaffolds were prepared by pore-causing agent leaching method, and then placed in xanthohumol solution for 24 hours to prepare xanthohumol-PLGA scaffolds (hereinafter referred to as drug-loaded scaffolds). The PLGA scaffolds and drug-loaded scaffolds were taken for general observation, the pore diameter of the scaffolds was measured by scanning electron microscope, the porosity was calculated by the drainage method, and the loading of xanthohumol on the scaffolds was verified by Fourier transform infrared (FTIR) spectrometer. Then the two scaffolds were co-cultured with RAW264.7 macrophages induced by lipopolysaccharide for 24 hours, and the expressions of inflammatory factors [interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α)] were detected by RT-PCR and Western blot to evaluate the anti-inflammatory properties in vitro of two scaffolds. Bone marrow mesenchymal stem cells (BMSCs) was obtained from bone marrow of a 6-month-old female healthy goat, cultured by adherent method, and passaged in vitro. The second passage cells were seeded on two scaffolds to construct BMSCs-scaffolds, and the cytocompatibility of scaffolds was observed by live/dead cell staining and cell counting kit 8 (CCK-8) assay. The BMSCs-scaffolds were cultured in vitro for 6 weeks, aiming to verify its feasibility of generating cartilage in vitro by gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, and biochemical analysis. Finally, the two kinds of BMSCs-scaffolds cultured in vitro for 6 weeks were implanted into the goat subcutaneously, respectively. After 4 weeks, gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, biochemical analysis, and RT-PCR were performed to comprehensively evaluate the anti-inflammatory effect in vivo and promotion of cartilage regeneration of the drug-loaded scaffolds. Results: The prepared drug-loaded scaffold had a white porous structure with abundant, continuous, and uniform pore structures. Compared with the PLGA scaffold, there was no significant difference in pore size and porosity ( P>0.05). FTIR spectrometer analysis showed that xanthohumol was successfully loaded to PLGA scaffolds. The in vitro results demonstrated that the gene and protein expressions of inflammatory cytokines (IL-1ß and TNF-α) in drug-loaded scaffold significantly decreased than those in PLGA scaffold ( P<0.05). With the prolongation of culture, the number of live cells increased significantly, and there was no significant difference between the two scaffolds ( P>0.05). The in vitro cartilage regeneration test indicated that the BMSCs-drug-loaded scaffolds displayed smooth and translucent appearance with yellow color after 6 weeks in vitro culture, and could basically maintained its original shape. The histological and immunohistochemical stainings revealed that the scaffolds displayed typical lacunar structure and cartilage-specific extracellular matrix. In addition, quantitative data revealed that the contents of glycosaminoglycan (GAG) and collagen type Ⅱ were not significantly different from BMSCs-PLGA scaffolds ( P>0.05). The evaluation of cartilage regeneration in vivo showed that the BMSCs-drug-loaded scaffolds basically maintained their pre-implantation shape and size at 4 weeks after implantation in goat, while the BMSCs-PLGA scaffolds were severely deformed. The BMSCs-drug-loaded scaffolds had typical cartilage lacuna structure and cartilage specific extracellular matrix, and no obvious inflammatory cells infiltration; while the BMSCs-PLGA scaffolds had a messy fibrous structure, showing obvious inflammatory response. The contents of cartilage-specific GAG and collagen type Ⅱ in BMSCs-drug-loaded scaffolds were significantly higher than those in BMSCs-PLGA scaffolds ( P<0.05); the relative gene expressions of IL-1ß and TNF-α were significantly lower than those in BMSCs-PLGA scaffolds ( P<0.05). Conclusion: The drug-loaded scaffolds have suitable pore size, porosity, cytocompatibility, and good anti-inflammatory properties, and can promote cartilage regeneration after implantation with BMSCs in goats.

Differences in semen quality, testicular histomorphology, fertility, reproductive hormone levels, and expression of candidate genes according to sperm motility in Beijing-You chickens.

Sperm motility is used to predict the semen quality and fertilization ability of breeder roosters. Indigenous chicken breeder roosters show variation in sperm motility, but less has been known about its relationship with other reproductive attributes. In this study, 40- to 44-wk-old Beijing-You breeder roosters with the high and low sperm motility phenotypes (n = 15 in each group) were identified and compared, with regard to their semen characteristics, reproductive hormone concentrations, testicular histomorphology, fertility capacity, and genes expression of hematopoietic prostaglandin D synthase (H-PGDS) and wingless-type MMTV integration site family, member 2 (WNT2). The low sperm motility roosters showed lower semen volume (P = 0.003) and sperm viability (P = 0.008). Like semen donors in the artificial insemination test, the low sperm motility roosters showed lower fertility than the high sperm motility roosters (P < 0.001). No difference in the serum concentration of follicle stimulating hormone (FSH, P = 0.347), luteinizing hormone (LH, P = 0.376), or testosterone (T, P = 0.068) was found between the high and low sperm motility roosters. Furthermore, among roosters with similar BW, the low sperm motility roosters had smaller testis mass (P = 0.037), seminiferous tubules diameter (P = 0.031), spermatogenic epithelium height (P = 0.001), and Johnsen score (P = 0.005) than the high sperm motility ones. Finally, the expression of both H-PGDS and WNT2, which are involved in the prostaglandin D2 and WNT signaling pathways, respectively, was low in the testis of the low sperm motility phenotype roosters (P < 0.05). Thus, the findings of the present study indicated that the testis of low sperm motility showed impaired testicular histomorphology as compared to normal; the BW, testis weight, and serum FSH, LH, and T levels are associated with sperm production, but not with sperm motility, and that low expression of H-PGDS and WNT2 may be related with the abnormal testicular morphology of low sperm motility birds which deserves further functional validation studies.

Identification and differential expression of microRNAs in the testis of chicken with high and low sperm motility.

Sperm motility is one of the most important indicators for evaluating roosters' fecundity. However, molecular regulation underlying chickens' sperm motility remains to be understood. MicroRNA (miRNA) plays an epigenetic role in reproduction. In this study, we compared the testicular miRNAs of Beijing-you roosters with high (HS) and low (LS) sperm motility using Illumina sequencing to try to understand the associated molecular regulation. Length distribution analysis showed that the dominant size of small RNAs detected in the testes of the chicken samples was 24-28 nt. In total, 518 known and 106 novel miRNAs were identified. A total of 23 miRNAs were found differentially expressed (P < 0.05, |log2fold change| ≥ 1) between the HS and LS groups, including 18 known and 5 novel miRNAs. Functional enrichment of the predicted target genes of the differentially expressed miRNAs indicated that these miRNAs were involved in the pathways of GnRH, MAPK and Wnt signaling. The miRNA-gene interaction network revealed two key candidate miRNA-gene pairs that might affect chicken sperm motility, viz, gga-miR-155/KCNA1 and gga-miR-7480-5p/AHI1. qPCR was then used to further validate their expressions. The results here provided a deep insight into the expressions of the miRNAs in the testes of chickens and suggested their roles in sperm motility regulation.

Single SNP- and pathway-based genome-wide association studies for beak deformity in chickens using high-density 600K SNP arrays.

BACKGROUND: Beak deformity, typically expressed as the crossing of upper and lower mandibles, is found in several indigenous chicken breeds, including the Beijing-You chickens studied here. Beak deformity severely impairs the birds' growth and welfare. Although previous studies shed some light on the genetic regulation of this complex trait, the genetic basis of this malformation remains incompletely understood. RESULTS: In this study, single SNP- and pathway-based genome-wide association studies (GWASs) were performed using ROADTRIPS and SNP ratio test (SRT), respectively. A total of 48 birds with deformed beaks (case) and 48 normal birds (control) were genotyped using Affymetrix 600 K HD genotyping arrays. As a result, 95 individuals and 429,539 SNPs were obtained after quality control. The P-value was corrected by a Bonferroni adjustment based on linkage disequilibrium pruning. The single SNP-based association study identified one associated SNP with 5% genome-wide significance and seven suggestively associated SNPs. Four high-confidence genes, LOC421892, TDRD3, RET, and STMN1, were identified as the most promising candidate genes underlying this complex trait in view of their positions, functions, and overlaps with previous studies. The pathway-based association study highlighted the association of six pathways with beak deformity, including the calcium signaling pathway. CONCLUSIONS: Potentially useful candidate genes and pathways for beak deformity were identified, which should be the subject of further functional characterization.

Analyses of Long Non-Coding RNA and mRNA profiling using RNA sequencing in chicken testis with extreme sperm motility.

Sperm motility is the most important indicator in evaluating roosters' fecundity. However, the genetic mechanisms underlying chicken sperm motility is not yet clear. Long non-coding RNA (lncRNA) play epigenetic roles in reproduction. In this study, RNA sequencing was employed to profile the testis transcriptome (lncRNA and mRNA) of six Beijing-you cocks divergent in sperm motility. In total, 2,597 lncRNAs were identified in the chicken testis, including 1,267 lincRNAs, 975 anti-sense lncRNAs, and 355 intronic lncRNAs. They shared similar features with previous studies. Of these lncRNAs, 124 were differentially expressed. Among 17,690 mRNAs detected in this study, 544 were differentially expressed, including a bunch of genes with known functions on sperm motility. GO annotation analysis revealed these genes were involved in ATP binding, cilium assembly, and oxidation-reduction processes. Integrating analysis of lncRNA and mRNA profiles predicted 10 lncRNA-gene pairs, including 8 co-regulated and 2 inversely-regulated pairs. To the best of our knowledge, this is the first genome-wide investigation of the lncRNAs in the chicken testis associated with sperm motility. Our results provided a catalog of chicken testis lncRNAs and genes worthy of further studies to understand their roles in cocks' reproductive performance regulation.

The pharmacokinetic and pharmacodynamic properties of site-specific pegylated genetically modified recombinant human interleukin-11 in normal and thrombocytopenic monkeys.

In order to improve the pharmacokinetic and pharmacodynamic properties of recombinant human interleukin-11 mutein (mIL-11) and to reduce the frequency of administration, we examined the feasibility of chemical modification of mIL-11 by methoxy polyethylene glycol succinimidyl carbonate (mPEG-SC). PEG-mIL-11 was prepared by a pH controlled amine specific method. Bioactivity of the protein was determined in a IL-11-dependent in vitro bioassay, its pharmacodynamic and pharmacokinetic properties were investigated by using normal and thrombocytopenic monkey models. N-terminus sequencing and peptide mapping analysis revealed that Lys33 is the PEGylated position for PEG-mIL-11. Bioactivity of PEG-mIL-11 assessed by B9-11 cell proliferation assay was comparable to that of mIL-11. More than 79-fold increase in area-under-the curve (AUC) and 26-fold increase in maximum plasma concentration (Cmax) was observed in pharmacokinetic analysis. Single dose administration of the PEG-mIL-11 induced blood platelets number increase and the effect duration were comparable to that of 7 to 10 consecutive daily administration of mIL-11 to the normal and thrombocytopenic monkey models. PEG-mIL-11 is a promising therapeutic for thrombocytopenia.

[Thrombocytopoietic Efficacy of Pegylated Recombinant Human Interleukin-11 Mutein in Myelosuppressed Mice].

OBJECTIVE: To evaluate the effect of PEGylated IL-11 mutein (PEG-mIL 11) with different dose or injection frequency on thrombocytopenia in myelosuppressed mice and to compare its effect with mIL-11, so as to provide reference data for clinical use. METHODS: Myelosuppressive model with thrombocyopenia was produced in BALB/c mice by whole body 60Co γ-ray irradiation in dose of administration 2.5 Gy followed by i.p. injections of carboplatin at 50 mg/kg. In study of injection frequency, 30 thrombocytopenic BALB/c mice were randomly divided into 5 groups: vehicle control group (once daily on d1, 4, 7), mIL-11 group [200 µg/(kg·d)×9 d], PEG-mIL 11 A group [111800 µg/(kg·d)×1 d (d 1)], PEG-mIL 11 B group 900 µg/(kg·d)×2 d (d 1,5), and PEG-mIL 11 C group [600 µg/(kg·d)×3 d (d 1,4,7)]. The route of administration is subcutaneous injection. The platelet counts were monitored in the subsequant 5 weeks. In study of dose administration, 100 thrombocytopenic BALB/c mice were randomly divided into 5 groups: vehicle control group (once daily on d1 and 5), mIL-11 group 200 µg/(kg·d)×9 d, and PEG-mIL 11 low-, mid-, and high-dose groups (200, 420 and 900 µg/(kg·d)×2 d, once a day on d1 and 5). The route of administration is subcutaneous injection. The platelet counts were monitored every 2-3 days in the subsequant 5 weeks, and CFU-Meg was determined on d 8 of the bone marrow cells collection. RESULTS: After 60Co irradition and carboplatin injection, Plt level decreased with time, and a >80% reduction was noted at nadir when comparing with baseline. In frequency of administration study, the platelet nadir of the 3 PEG-mIL 11 groups were significantly higher than that of vehicle control and mIL-11 groups (P<0.05), while no significant difference was noted among the 3 groups of different administration frequences; In dose level study, the reduction of Plt count at the nadir in the 3 PEG-mIL 11 groups was significantly less than that of vehicle control and mIL-11 groups (P<0.05). And a rapid recovery of Plt count was found in the PEG-mIL 11 groups with a dose-dependent increase of Plt count on d 10. A lower reduction and a rapider recovery of RBC was also found in the PEG-mIL 11 groups. No significant effect on WBC was found for all the treatment groups. An increase in CFU-Meg was observed in PEG-mIL 11 and mIL-11 groups, with higher CFU-Meg in PEG-mIL 11 groups. CONCLUSION: A preventive effect of PEG-mIL 11 on thrombocytopenia in myelosuppressed mice has been confirmed. In comparison with mIL-11, a better effect of PEG-mIL 11 is obtained under lower dose frequency, indicating a better compliance of the treatment regimen, and providing a foundation for developing a long-acting preparation of rhIL-11.

[Expression of laminin 5 in rat skin wounds regulated by recombinant thymosin beta4].

OBJECTIVE: To explore the mechanism of recombinant thymosin beta4 (Tbeta4) accelerating skin wound healing in rats by regulating laminin 5 expression. METHODS: Two full thickness 8 mm punch wounds were made at the costovertebral angle on dorsal surface of each adult male rats weighing 200-250 g. Sixty rats were randomized into the control group (n = 15) and the experimental group (n = 45), which was subdivided into low, medium and high dose groups (n = 15). Tbeta4 was applied topically at 2, 6, 18 microg in 50 microL PBS for every 12 hours after model making in the experimental group. The identical amounts of phosphate buffered saline was applied in the control group. Wound healing was observed after model making and immunohistochemical observation was conducted 2, 4 and 7 days after operation. RESULTS: Seven days after operation, wound contracted obviously and most of the wounds connected well with the margin. In the control group, low dose group, medium dose group and high dose group, the wound healing rate were 7.67% +/- 5.46%, 29.01% +/- 7.43%, 26.54% +/- 11.49% and 10.39% +/- 3.96% respectively 2 days after operation; 28.16% +/- 13.76%, 37.99% +/- 13.05%, 42.00% +/- 9.56% and 39.58% +/- 12.74% respectively 4 days after operation; 59.08% +/- 19.02%, 64.15% +/- 17.92%, 77.39% +/- 8.45% and 69.78% +/- 8.45% respectively 7 days after operation. At 2 days after operation, significant differences were notified in healing rats between 3 sub-experimental groups and the control group (P < 0.05). Immunohistochemistry staining showed that there was a little more positive expression of laminin 5 2 days after operation that beneficial to promote the proliferation and differentiation of cell in every group, including positive cells and ECM. But in medium group there was fewer expression, only at the borderline and bottom of the wound, while the expression significantly increased 4 days after operation (P < 0.05) and there was a relative high expression 7 days after operation (P < 0.01). CONCLUSION: Tbeta4 can inhibit the expression of laminin 5 early, and then up-regulate laminin 5 expression to moderate the reformation of ECM, promote the migration of epidemic cell and accelerate skin wound healing.