Fiber-reinforced gelatin/ß-cyclodextrin hydrogels loaded with platelet-rich plasma-derived exosomes for diabetic wound healing.

Diabetic complications with high-glucose status (HGS) cause the dysregulated autophagy and excessive apoptosis of multiple-type cells, leading to the difficulty in wound self-healing. Herein, we firstly developed fiber-reinforced gelatin (GEL)/ß-cyclodextrin (ß-CD) therapeutic hydrogels by the modification of platelet-rich plasma exosomes (PRP-EXOs). The GEL fibers that were uniformly dispersed within the GEL/ß-CD hydrogels remarkably enhanced the compression strengths and viscoelasticity. The PRP-EXOs were encapsulated in the hydrogels via the covalent crosslinking between the PRP-EXOs and genipin. The diabetic rat models demonstrated that the GEL/ß-CD hydrogels and PRP-EXOs cooperatively promoted diabetic wound healing. On the one hand, the GEL/ß-CD hydrogels provided the biocompatible microenvironments and active components for cell adhesion, proliferation and skin tissue regeneration. On the other hand, the PRP-EXOs in the therapeutic hydrogels significantly activated the autophagy and inhibited the apoptosis of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs). The activation of autophagy and inhibition of apoptosis in HUVECs and HSFs induced the blood vessel creation, collagen formation and re-epithelialization. Taken together, this work proved that the incorporation of PRP-EXOs in a wound dressing was an effective strategy to regulate autophagy and apoptosis, and provide a novel therapeutic platform for diabetic wound healing.

Repair of Spinal Cord Injury by Inhibition of PLK4 Expression Through Local Delivery of siRNA-Loaded Nanoparticles.

Polo-like kinase 4 (PLK4) is one of the key regulators of centrosomal replication. However, its role and mechanism in spinal cord injury (SCI) are still unclear. The SCI model on rats was constructed and the expression and localization of PLK4 in the spinal cord are analyzed with Western blot and immunofluorescence, respectively. Then the specific siRNAs were encapsulated in nanoparticles for the inhibition of PLK4 expression. Afterward, the role of PLK4 on astrocytes was investigated by knocking down its expression in the primary astrocytes. Moreover, siRNA-loaded nanoparticles were injected into the injured spinal cord of rats, and the motor function recovery of rats after SCI was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale method. Notably, the siRNA-loaded nanoparticles effectively transfect primary astrocytes and significantly inhibit PLK4 expression, together with the expression of PCNA with significance. After treatment, restoration of the motor function following SCI was significantly improved in the PLK4 knockdown group compared with the control group. Therefore, we speculate that inhibition of Plk4 may inhibit the proliferation of astrocytes and decrease the inflammatory response mediated by astrocytes, so as to promote the functional recovery of SCI. In conclusion, inhibition of PLK4 expression via siRNA-loaded nanoparticles may be a potential treatment for SCI.

[Research progress of acupuncture in enhanced recovery after surgery].

The concept of enhanced recovery after surgery (ERAS) is based on evidence-based medicine. By optimizing the treatment measures, the stress response of the body is reduced to meet the body's metabolic changes during the perioperative period, so as to achieve the purpose of accelerating recovery. In the perioperative period, acupuncture can relieve patients' preoperative anxiety, shorten fasting time, reduce the amount of anesthetics, protect organ function, reduce postoperative pain, reduce postoperative nausea and vomiting, and speed up postoperative recovery. The addition of acupuncture intervention in ERAS would provide newmethods and new ideas for the construction of ERAS. But at present, the application of acupuncture in ERAS has problems such as lacking of overall consideration and insufficient standardization, etc.

Transcription factor EB is involved in autophagy-mediated chemoresistance to doxorubicin in human cancer cells.

Transcription factor EB (TFEB) is a master regulator of autophagy activity and lysosomal biogenesis, but its role in autophagy-mediated cell survival and chemotherapy resistance is not completely understood. In this study, we explored whether TFEB played an important role in autophagy-mediated chemotherapy resistance in human cancer LoVo and HeLa cells in vitro. Treatment of human colon cancer LoVo cells with doxorubicin (0.5 µmol/L) induced autophagy activation and nuclear translocation of TFEB, which resulted from inactivation of the mTOR pathway. In both LoVo and HeLa cells, overexpression of TFEB enhanced doxorubicin-induced autophagy activation and significantly decreased doxorubicin-induced cell death, whereas knockdown of TFEB with small interfering RNA blocked doxorubicin-induced autophagy and significantly enhanced the cytotoxicity of doxorubicin. In LoVo cells, autophagy inhibition by 3-methyladenine (3-MA) or knockdown of autophagy-related gene Atg5 increased cell death in response to doxorubicin, and abolished TFEB overexpression-induced chemotherapy resistance, suggesting that the inhibition of autophagy made cancer cells more sensitive to doxorubicin. The results demonstrate that TFEB-mediated autophagy activation decreases the sensitivity of cancer cells to doxorubicin.

Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis.

AIMS: Point-of-care genetic analysis may require polymerase chain reaction (PCR) to be carried out on whole blood. However, human blood contains natural inhibitors of PCR such as hemoglobin, immunoglobulin G, lactoferrin, and proteases, as well as anticoagulant agents, including EDTA and heparin that can reduce whole blood PCR efficiency. Our purpose was to develop a highly specific, direct whole blood single-nucleotide polymorphism (SNP) analysis method based on allele-specific (AS) PCR that is mediated by Pfu DNA polymerase and phosphorothioate-modified AS primers. RESULTS: At high Mg(2+) concentrations, Pfu DNA polymerase efficiently amplified genomic DNA in a reaction solution containing up to 14% whole blood. Among the three anticoagulants tested, Pfu DNA polymerase showed the highest activity with sodium citrate. Meanwhile, Triton X-100 and betaine inhibited Pfu DNA polymerase activity in whole blood PCR, whereas trehalose had virtually no effect. These findings provided for the development of a low-cost, simple, and fast direct whole blood genotyping method that uses Pfu DNA polymerase combined with phosphorothioate AS primers for CYP2C9*3 and VKORC1(-1639) loci. CONCLUSIONS: With its high DNA amplification efficiency and tolerance of various blood conditions, Pfu DNA polymerase can be used in clinical laboratories to analyze SNPs in whole blood samples.

Sestrin2 Protects Dopaminergic Cells against Rotenone Toxicity through AMPK-Dependent Autophagy Activation.

Dysfunction of the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS) was thought to be an important pathogenic mechanism in synuclein pathology and Parkinson's disease (PD). In the present study, we investigated the role of sestrin2 in autophagic degradation of α-synuclein and preservation of cell viability in a rotenone-induced cellular model of PD. We speculated that AMP-activated protein kinase (AMPK) was involved in regulation of autophagy and protection of dopaminergic cells against rotenone toxicity by sestrin2. The results showed that both the mRNA and protein levels of sestrin2 were increased in a TP53-dependent manner in Mes 23.5 cells after treatment with rotenone. Genetic knockdown of sestrin2 compromised the autophagy induction in response to rotenone, while overexpression of sestrin2 increased the basal autophagy activity. Sestrin2 presumably enhanced autophagy in an AMPK-dependent fashion, as sestrin2 overexpression activated AMPK, and genetic knockdown of AMPK abrogated autophagy induction by rotenone. Restoration of AMPK activity by metformin after sestrin2 knockdown recovered the autophagy activity. Sestrin2 overexpression ameliorated α-synuclein accumulation, inhibited caspase 3 activation, and reduced the cytotoxicity of rotenone. These results suggest that sestrin2 upregulation attempts to maintain autophagy activity and suppress rotenone cytotoxicity through activation of AMPK, and that sestrin2 exerts a protective effect on dopaminergic cells.

[Comparison of clinical outcomes between unilateral fixation fusion and minimally invasive spine transforaminal lumbar interbody fusion in treating lumbar disc herniation].

OBJECTIVE: To compare the short-term clinical outcome between unilateral fixation fusion (ULF) and minimally invasive spine transforaminal lumbar interbody fusion (MIS-TLIF) in treating lumbar disc herniation (LDH). METHODS: The clinical data of 39 patients with LDH were retrospectively analyzed from June 2008 to March 2013. There was 22 males and 17 females, aged from 45 to 75 years old with an average of 56.9 years. Therer were 3 cases in L3,4, 15 cases in L4,5, 21 cases in L5S1. Among them, 21 patients underwent unilateral fixation fusion (ULF group) and 18 underwent minimally invasive spine transforaminal lumbar interbody fusion (MIS-TLIF group). Operation time, blood loss, the times of radiographic exposure and hospital stay were noted and compared between two groups. Radiograph informations were regularily accessed and VAS, ODI scores were recorded at 3 days and 3, 6, 12 months after operation, respectively. According to modified Macnab criteria, the clinical effects were evaluated at final follow-up. RESULTS: All operations were successful without severe complications. The averaged operative time and the times of radiographic exposure in ULF group [(95 ± 25) min and (4.2 ± 0.4) times] were less than that of MIS-TLIF group [(120 ± 35) min and (10.1 ± 3.9) times] (P < 0.05). But, the mean blood loss and hospital stay in MIS-TLIF group [(75 ± 45) ml and (7.2 ± 2.2)d ]were less than that of ULF group [(165 ± 60) ml and (11.0 ± 3.7) d] (P < 0.01). All patients were followed up from 12 to 45 months with an average of 29.5 months. The VAS and ODI score had significantly improved during the follow-up and no significant differences were found between two groups at the same time point (P > 0.05). The postoperative radiographs showed internal fixation position was good. And all patients obtained bone fusion by CT scan at 1 year after operation. There was no significant differences in modified Macnab criteria between two groups at the latest follow-up (P > 0.05). CONCLUSION: Favorable short-term clinical effects can be achieved in suitable LDH patients with ULF or MIS-TLIF surgical procedures.

Hydroxyapatite coatings with oriented nanoplate arrays: synthesis, formation mechanism and cytocompatibility.

Hydroxyapatite (HA) is the main inorganic constituent of natural bones and teeth with c-axis orientation and a(b)-axis orientation, respectively. Designing HA coatings (HACs) with specific orientation and morphology is an important strategy to improve their biological properties. Herein, we report, for the first time, the hydrothermal synthesis of HACs with oriented nanoplate arrays according to the following steps: (i) deposition of brushite/chitosan coatings (BCCs) on Ti6Al4V substrates; and (ii) transformation of HACs with oriented nanoplate arrays from BCCs after hydrothermal treatment with alkaline solutions. After soaking the BCCs in a NaOH solution under hydrothermal conditions, the Ca2+ and PO4 3- ions are released from the coatings because of the dissolution reaction of brushite, and they react with OH- ions to form HA nanoplates. Interestingly, these HA nanoplates with a preferential c-plane orientation are perpendicular to the coating surfaces. Hydrothermal reaction time and Ca/P ratio of BCCs have great effects on the morphologies of HA nanoplates. On increasing the reaction time from 3 h to 3 days or decreasing the Ca/P ratio from 2.0 to 1.0, the widths (or lengths) of HA nanoplates increase gradually. Simulated body fluid immersion (SBF) tests reveal that the HACs with oriented nanoplate arrays can promote the formation of apatite on the surfaces, suggesting their good in vitro bioactivity. Moreover, human bone marrow stromal cells (hBMSCs) have been used as cell models to investigate cytocompatibility of the HACs. The hBMSCs on the HACs have better cell adhesion, spreading, proliferation and osteogenic differentiation than those on Ti6Al4V substrates because the HACs are similar to the minerals of human hard tissues in chemical composition, morphology and crystallographic orientation. Therefore, HACs with oriented nanoplate arrays have great potential for use as implants of human hard tissues.

Hybrid nanostructured hydroxyapatite-chitosan composite scaffold: bioinspired fabrication, mechanical properties and biological properties.

The fabrication of bone scaffolds with interconnected porous structure, adequate mechanical properties, excellent biocompatibility and osteoinductivity presents a great challenge. Herein, a hybrid nanostructured hydroxyapatite-chitosan (HA-CS) composite scaffold has been fabricated according to the following steps: (i) the deposition of brushite-CS on a CS fibre porous scaffold by a dip-coating method; and (ii) the formation of a hybrid nanostructured HA-CS composite scaffold by the in situ conversion of brushite to HA using a bioinspired mineralization process. The hybrid HA-CS composite scaffold possesses three-dimensional (3D) interconnected pores with pore sizes of 30-80 µm. The HA rods with a length of ∼200 nm and width of ∼50 nm are perpendicularly oriented to the CS fibres. Interestingly, the abovementioned HA rods are composed of many smaller nanorods with a length of ∼40 nm and width of ∼10 nm oriented along the c-axis. The hybrid nanostructured HA-CS composite scaffold exhibits good mechanical properties with a compression strength of 9.41 ± 1.63 MPa and an elastic modulus of 0.17 ± 0.02 GPa, which are well-matched to those of trabecular bone. The influences of the hybrid HA-CS composite scaffold on cells have been investigated using human bone marrow stem cells (hBMSCs) as cell model and the CS fibre porous scaffold as the control sample. The hybrid HA-CS composite scaffold not only supports the adhesion and proliferation of hBMSCs, but also improves the osteoinductivity. The alkaline phosphatase activity and mineralization deposition on the hybrid HA-CS composite scaffold are higher than those on the CS fibre porous scaffold. Moreover, the hybrid HA-CS composite scaffold can promote the formation of new bone in rat calvarial defects as compared with the CS fibre porous scaffold. The excellent biocompatibility, osteoinductivity and mechanical properties suggest that the hybrid nanostructured HA-CS composite scaffold has great potential for bone tissue engineering.

Dimethyloxaloylglycine improves angiogenic activity of bone marrow stromal cells in the tissue-engineered bone.

One of the big challenges in tissue engineering for treating large bone defects is to promote the angiogenesis of the tissue-engineered bone. Hypoxia inducible factor-1α (HIF-1α) plays an important role in angiogenesis-osteogenesis coupling during bone regeneration, and can activate a broad array of angiogenic factors. Dimethyloxaloylglycine (DMOG) can activate HIF-1α expression in cells at normal oxygen tension. In this study, we explored the effect of DMOG on the angiogenic activity of bone mesenchymal stem cells (BMSCs) in the tissue-engineered bone. The effect of different concentrations of DMOG on HIF-1a expression in BMSCs was detected with western blotting, and the mRNA expression and secretion of related angiogenic factors in DMOG-treated BMSCs were respectively analyzed using qRT-PCR and enzyme linked immunosorbent assay. The tissue-engineered bone constructed with ß-tricalcium phosphate (ß-TCP) and DMOG-treated BMSCs were implanted into the critical-sized calvarial defects to test the effectiveness of DMOG in improving the angiogenic activity of BMSCs in the tissue-engineered bone. The results showed DMOG significantly enhanced the mRNA expression and secretion of related angiogenic factors in BMSCs by activating the expression of HIF-1α. More newly formed blood vessels were observed in the group treated with ß-TCP and DMOG-treated BMSCs than in other groups. And there were also more bone regeneration in the group treated with ß-TCP and DMOG-treated BMSCs. Therefore, we believed DMOG could enhance the angiogenic activity of BMSCs by activating the expression of HIF-1α, thereby improve the angiogenesis of the tissue-engineered bone and its bone healing capacity.

Biological characteristics of human-urine-derived stem cells: potential for cell-based therapy in neurology.

Stem cells in human urine have gained attention in recent years; however, urine-derived stem cells (USCs) are far from being well elucidated. In this study, we compared the biological characteristics of USCs with adipose-derived stem cells (ASCs) and investigated whether USCs could serve as a potential cell source for neural tissue engineering. USCs were isolated from voided urine with a modified culture medium. Through a series of experiments, we examined the growth rate, surface antigens, and differentiation potential of USCs, and compared them with ASCs. USCs showed robust proliferation ability. After serial propagation, USCs retained normal karyotypes. Cell surface antigen expression of USCs was similar to ASCs. With lineage-specific induction factors, USCs could differentiate toward the osteogenic, chondrogenic, adipogenic, and neurogenic lineages. To assess the ability of USCs to survive, differentiate, and migrate, they were seeded onto hydrogel scaffold and transplanted into rat brain. The results showed that USCs were able to survive in the lesion site, migrate to other areas, and express proteins that were associated with neural phenotypes. The results of our study demonstrate that USCs possess similar biological characteristics with ASCs and have multilineage differentiation potential. Moreover USCs can differentiate to neuron-like cells in rat brain. The present study shows that USCs are a promising cell source for tissue engineering and regenerative medicine.