Oscillating Fluid Flow Activated Osteocyte Lysate-Based Hydrogel for Regulating Osteoblast/Osteoclast Homeostasis to Enhance Bone Repair.

As major regulators on bone formation/resorption in response to mechanical stimuli, osteocytes have shown great promise for restoring bone injury. However, due to the unmanageable and unabiding cell functions in unloading or diseased environments, the efficacy of osteogenic induction by osteocytes has been enormously limited. Herein, a facile method of oscillating fluid flow (OFF) loading for cell culture is reported, which enables osteocytes to initiate only osteogenesis and not the osteolysis process. After OFF loading, multiple and sufficient soluble mediators are produced in osteocytes, and the collected osteocyte lysates invariably induce robust osteoblastic differentiation and proliferation while restraining osteoclast generation and activity under unloading or pathological conditions. Mechanistic studies confirm that elevated glycolysis and activation of the ERK1/2 and Wnt/ß-catenin pathways are the major contributors to the initiation of osteoinduction functions induced by osteocytes. Moreover, an osteocyte lysate-based hydrogel is designed to establish a stockpile of "active osteocytes" to sustainably deliver bioactive proteins, resulting in accelerated healing through regulation of endogenous osteoblast/osteoclast homeostasis.

Targeting hexokinase 2 increases the sensitivity of oxaliplatin by Twist1 in colorectal cancer.

Colorectal cancer (CRC) is the third most malignant tumour worldwide, with high mortality and recurrence. Chemoresistance is one of the main factors leading to metastasis and poor prognosis in advanced CRC patients. By analysing the Gene Expression Omnibus data set, we found higher hexokinase 2 (HK2) expression levels in patients with metastatic CRC than in those with primary CRC. Moreover, we observed higher enrichment in oxaliplatin resistance-related gene sets in metastatic CRC than in primary CRC. However, the underlying relationship has not yet been elucidated. In our study, HK2 expression was significantly elevated in CRC patients. Gene set enrichment analysis (GSEA) revealed multi-drug resistance and epithelial-mesenchymal transition (EMT) pathways related to high HK2 expression. Our results showed that knockdown of HK2 significantly inhibited vimentin and Twist1 expression and promoted TJP1 and E-cadherin expression in CRC cells. Additionally, transcriptional and enzymatic inhibition of HK2 by 3-bromopyruvate (3-bp) impaired oxaliplatin resistance in vitro and in vivo. Mechanistically, HK2 interacts with and stabilized Twist1 by preventing its ubiquitin-mediated degradation, which is related to oxaliplatin resistance, in CRC cells. Overexpression of Twist1 reduced the apoptosis rate by HK2 knockdown in CRC cells. Collectively, we discovered that HK2 is a crucial regulator that mediates oxaliplatin resistance through Twist1. These findings identify HK2 and Twist1 as promising drug targets for CRC chemoresistance.

Injectable and bioactive methylcellulose hydrogel carrying bone mesenchymal stem cells as a filler for critical-size defects with enhanced bone regeneration.

As a soluble cellulose derivative, methylcellulose (MC), can be used to construct thermosensitive hydrogels. However, a pure MC gel is generally considered an inert material that is inadequate for cell survival. We adopted an environmentally friendly method to fabricate a nano-hydroxyapatite (nHA) hybrid MC hydrogel. Rheology results showed that the addition of nHA increased the gelation temperature. Furthermore, the live/dead assay confirmed that the addition of nHA improved the survival of bone marrow-derived mesenchymal stem cells (BMSCs) inside the gel. In addition, ARS staining indicated that the presence of nHA stimulated osteogenic differentiation. Finally, in vivo cranial defect experiments showed improved remediation efficiency when using the nHA hybrid MC hydrogel to carry BMSCs.

Hydrophobic IR780 loaded sericin nanomicelles for phototherapy with enhanced antitumor efficiency.

The near-infrared dye, IR780 iodide, has been utilized in photodynamic therapy (PDT) and photothermal therapy (PTT). However, the hydrophobicity and photosensitivity of IR780 limit its further applications in biomedical fields. Herein, the hydrophilic sericin was modified with hydrophobic cholesterol to form an amphiphilic macromolecular conjugate (Ser-Chol). The tumor-targeting agent, folic acid (FA), was further linked to the conjugate (FA-Ser-Chol). The IR780 could be encapsulated into such amphiphilic macromolecule to form stable micelles (FA-Ser-Chol/IR780) by self-assembly, and the solubility and photo-stability of IR780 were greatly improved. The FA-Ser-Chol/IR780 micelles could be efficiently absorbed by FA-positive gastric cancer cells (BGC-823) through FA receptors, while the uptake micelles showed remarkable PDT and PTT cytotoxicity towards BGC-823 cells under laser irradiation of 808 nm. Therefore, FA-Ser-Chol micelles may serve as a promising IR780 carrier for PDT and PTT therapy.

Vitamin B12-conjugated sericin micelles for targeting CD320-overexpressed gastric cancer and reversing drug resistance.

AIM: Our previous research has introduced sericin micelles to reverse drug resistance. However, these micelles could not selectively bind to gastric cancer (GC) cells. We developed vitamin B12 (VB12) conjugated sericin micelles for targeted GC therapy. MATERIALS & METHODS: We used VB12, sericin, synthetic poly(γ-benzyl-L-glutamate) (PBLG) and paclitaxel (PTX) to develop VB12-conjugated and PTX-loaded micelles (VB12-sericin-PBLG-PTX). Then we explored their physicochemical properties, cellular uptake and antitumor mechanism. RESULTS: VB12-sericin-PBLG-PTX micelles were proved to be of appropriate particle size, have good dispersion and are bio-safe. Following transcobalamin II (CD320)-receptor-mediated endocytosis, these swallowed micelles with GC-targeting and enhanced cellular uptake abilities, alter mitochondrial transmembrane potential/apoptosis pathway and reverse drug resistance. CONCLUSION: VB12-sericin-PBLG-PTX micelles are promising materials for GC-targeted clinical applications.

[Effect of poly (L-lysine) modified silk fibroin film on the growth and differentiation of neural stem cells].

In order to provide a basic theory for the materials of repairing central nervous system injury, we have studied the growth and differentiation of neural stem cells (NSCs) on poly (L-lysine) modified silk fibroin film. First, we used poly (L-lysine) to modify silk fibroin film and confirmed by UV-vis and 1H NMR spectra. Then NSCs were isolated and seeded on the silk fibroin film (Silk group), poly (L-lysine) (PLL group) and poly (L-lysine) modified Silk fibroin film (Silk-PIL group). The proliferation of NSCs was evaluated by Cell Counting Kit-8 (CCK-8) assay on days 1, 3, 5 and 7 after seeding. Immunofluorescence was used to analyze the differentiation of NSCs at the 7th day. The levels of apoptosis were detected by Western blotting and TUNEL. The mRNA level of brain derived neurotrophic factor (BDNF) was identified by real-time PCR. UV-vis and 1H NMR spectra confirmed that poly (L-lysine) was successfully grafted onto the silk fibroin film. From the 3rd day after seeding to the 7th day, the CCK-8 test showed that proliferation rate of NSCs in the Silk-PIL was significantly higher than Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Immunofluorescence staining displayed that more NSCs in Silk-PIL group were differentiated into neuron compared with Silk group (P<0.05), however, there was no significant difference compared with PLL group (P>0.05). The number of NSCs differentiated into astrocytes was not significantly different between the three groups. Western blotting and TUNEL test presented that the degree of apoptosis of NSCs in the Silk-PIL group was significantly lower than Silk group (P<0.05). RT-PCR exhibited that mRNA level of brain derived neurotrophic factor (BDNF) of NSCs was higher in Silk-PIL group compared with Silk group (P<0.05) but had no significant difference compared with PLL group (P>0.05). Thus, poly (L-lysine) modified silk fibroin film could promote the proliferation of NSCs and reduce NSCs apoptosis. Furthermore, it also can enhance the differentiation of NSCs into neurons. It is expected to become a new type of tissue engineering scaffold carrying NSCs to repair central nervous system injury.

Sericin nanomicelles with enhanced cellular uptake and pH-triggered release of doxorubicin reverse cancer drug resistance.

Drug resistance is the major challenge facing cancer chemotherapy and nanoscale delivery systems based on natural materials, such as sericin, are a promising means of overcoming drug resistance. Yet, no attempt of introducing synthetic poly(γ-benzyl-L-glutamate) (PBLG) onto sericin polypeptide to fabricate a facile biocompatible and biodegradable micelle has been tried. Here, we prepared a polypeptide-based amphiphilic polymer containing hydrophilic sericin polypeptide backbone and PBLG side chains via ring-opening polymerization (ROP) strategy. The introduction of PBLG side chains remarkably enhances the stability of sericin micelles in water. Meanwhile, the micelles exhibited a high loading capacity and pH-responsive release ability for antitumor drug doxorubicin (DOX), called sericin-PBLG-DOX. Owing to the excellent cell membrane penetration of sericin-PBLG, the cellular uptake of DOX when loaded into micelles was improved. Subsequently, sericin-PBLG-DOX was transferred into perinuclear lysosomes, where the release rate of DOX was accelerated. Compared to the same dose of DOX, sericin-PBLG-DOX could induce a more efficient anti-tumor effect both in vitro and in vivo, and these micelles have promise for future clinical applications in overcoming cancer drug resistance with good biosafety, enhanced cellular uptake, pH-triggered drug release, efficient anti-tumor effects, and minimized systemic toxicity.

Sustained dual release of placental growth factor-2 and bone morphogenic protein-2 from heparin-based nanocomplexes for direct osteogenesis.

OBJECTIVE: To compare the direct osteogenic effect between placental growth factor-2 (PlGF-2) and bone morphogenic protein-2 (BMP-2). METHODS: Three groups of PlGF-2/BMP-2-loaded heparin-N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) nanocomplexes were prepared: those with 0.5 µg PlGF-2; with 1.0 µg BMP-2; and with 0.5 µg PlGF-2 combined with 1.0 µg BMP-2. The loading efficiencies and release profiles of these growth factors (GFs) in this nanocomplex system were quantified using enzyme-linked immunosorbent assay, their biological activities were evaluated using cell counting kit-8, cell morphology, and cell number counting assays, and their osteogenic activities were quantified using alkaline phosphatase and Alizarin Red S staining assays. RESULTS: The loading efficiencies were more than 99% for the nanocomplexes loaded with just PlGF-2 and for those loaded with both PlGF-2 and BMP-2. For the nanocomplex loaded with just BMP-2, the loading efficiency was more than 97%. About 83%-84% of PlGF-2 and 89%-91% of BMP-2 were stably retained on the nanocomplexes for at least 21 days. In in vitro biological assays, PlGF-2 exhibited osteogenic effects comparable to those of BMP-2 despite its dose in the experiments being lower than that of BMP-2. Moreover, the results implied that heparin-based nanocomplexes encapsulating two GFs have enhanced potential in the enhancement of osteoblast function. CONCLUSION: PlGF-2-loaded heparin-HTCC nanocomplexes may constitute a promising system for bone regeneration. Moreover, the dual delivery of PlGF-2 and BMP-2 appears to have greater potential in bone tissue regeneration than the delivery of either GFs alone.

A systematic computational study of the reactions of HO2 with RO2: the HO2 + CH2ClO2, CHCl2O2, and CCl3O2 reactions.

Potential energy surfaces for the reactions of HO(2) with CH(2)ClO(2), CHCl(2)O(2), and CCl(3)O(2) have been calculated using coupled cluster theory and density functional theory (B3LYP). It is revealed that all the reactions take place on both singlet and triplet surfaces. Potential wells exist in the entrance channels for both surfaces. The reaction mechanism on the triplet surface is simple, including hydrogen abstraction and S(N)2-type displacement. The reaction mechanism on the singlet surface is more complicated. Interestingly, the corresponding transition states prefer to be 4-, 5-, or 7-member-ring structures. For the HO(2) + CH(2)ClO(2) reaction, there are two major product channels, viz., the formation of CH(2)ClOOH + O(2) via hydrogen abstraction on the triplet surface and the formation of CHClO + OH + HO(2) via a 5-member-ring transition state. Meanwhile, two O(3)-forming channels, namely, CH(2)O + HCl + O(3) and CH(2)ClOH + O(3) might be competitive at elevated temperatures. The HO(2) + CHCl(2)O(2) reaction has a mechanism similar to that of the HO(2) + CH(2)ClO(2) reaction. For the HO(2) + CCl(3)O(2) reaction, the formation of CCl(3)O(2)H + O(2) is the dominant channel. The Cl-substitution effect on the geometries, barriers, and heats of reaction is discussed. In addition, the unimolecular decomposition of the excited ROOH (e.g., CH(2)ClOOH, CHCl(2)OOH, and CCl(3)OOH) molecules has been investigated. The implication of the present mechanisms in atmospheric chemistry is discussed in comparison with the experimental measurements.

Effects of hepatitis B virus infection on human sperm chromosomes.

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients. METHODS: Sperm chromosomes of 14 tested subjects (5 healthy controls, 9 patients with HBV infection, including 1 with acute hepatitis B, 2 with chronic active hepatitis B, 4 with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8 %, 33/223) was significantly higher than that in the control group (4.3 %, 5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4 signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random. CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.