Suppressing CHD1L reduces the proliferation and chemoresistance in osteosarcoma.

Osteosarcoma (OS) is the most common bone malignant tumor. However, the genetic basis of OS pathogenesis is still not understood, and occurrence of chemo-resistance is a major reason for the high morbidity of OS patients. Recently, chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) has been identified as a gene related to malignant tumor progression. Unfortunately, its effects on OS development and drug resistance are still not understood. In the study, we attempted to investigate the effects of CHD1L on tumorigenesis and chemoresistance in OS. We found that CHD1L expression was markedly up-regulated in OS samples, especially in cisplatin (cDDP)-resistant patients. We also showed that OS cells with CHD1L knockdown were more sensitive to cDDP treatment with lower IC50 values. In addition, we found that CHD1L deletion markedly reduced cell proliferation and induced apoptosis in OS cells with cDDP resistance. Moreover, the properties of cancer stem cells were highly suppressed in cDDP-resistant OS cells following CHD1L knockdown. Furthermore, multidrug resistance protein 1 (MDR-1) expression levels were dramatically decreased in OS cells with cDDP resistance when CHD1L was suppressed. Functional analysis indicated that CHD1L knockdown clearly restrained the activation of ERK1/2, protein kinase B (AKT) and NF-κB signaling pathways in cDDP-resistant OS cells. Consistently, animal experiments suggested that CHD1L suppression mitigated cDDP resistance in the generated in vivo xenografts. Collectively, CHD1L could modulate chemoresistance of OS cells to cDDP, and thus may be inspiring findings for overcoming drug resistance in OS.

Doubly crosslinked biodegradable hydrogels based on gellan gum and chitosan for drug delivery and wound dressing.

Biopolymer-based hydrogels with sustained drug release capability and antibacterial activity have exhibited great potential in clinical application in drug delivery and wound healing. In this study, a new type of composite wound dressing hydrogel aiming at avoiding wound infection was developed through embedding drug loaded gellan gum microspheres (GMs) into a doubly crosslinked hydrogel, which was constructed by Schiff-base crosslinking of oxidized gellan gum (OG) (pre-crosslinked by calcium ion) and carboxymethyl chitosan (CMCS). The gelation time, swelling index, degradation rate and mechanical properties of the blank hydrogel was optimized by varying the ratios of CMCS/OG (w/w) with fixed OG/calcium (w/w) ratio. The best overall performance of the hydrogel was obtained when CMCS/OG is 16/7 (w/w), with a 139 s gelation time, swelling index remained above 30 after swelling equilibrium, 100.5% degradation rate on the seventh day, and 8.8 KPa compressive modulus. After being embedded with cargo-loaded GMs, the aforementioned performance of the blank hydrogel was improved, and the sustained release of cargoes (antibacterial drugs, tetracycline hydrochloride and silver sulfadiazine) was observed. Moreover, the excellent antibacterial activity of the composite hydrogel was also demonstrated in vitro. These results support the bioactive composite hydrogel can be employed as a promising injectable scaffold for promoting wound regeneration and drug delivery.

MicroRNA-150 functions as a tumor suppressor and sensitizes osteosarcoma to doxorubicin-induced apoptosis by targeting RUNX2.

Osteosarcoma (OS) is the most common form of bone malignancy in children and adolescents. MicroRNAs (miRNAs) have been associated with the development and progression of OS. In the present study, reverse transcription-quantitative PCR, western blotting, Cell Counting Kit-8, luciferase and Transwell assays were performed to investigate the biological function of microRNA-150 (miR-150) in OS. The results revealed that miR-150 was significantly downregulated in OS cell lines (HOS, SAOS2, MG-63 and U2OS) in comparison with the normal osteoblast cells (hFOB1.19). Overexpression of miR-150 significantly inhibited cell proliferation in OS cells. miR-150 could sensitize OS cells to chemotherapy treatment of doxorubicin. Runt-related transcription factor 2 (RUNX2) was identified as a target gene of miR-150. RUNX2 knockdown exhibited similar inhibitory effects on both OS cell proliferation and chemotherapy sensitivity. Restoration of RUNX2 reversed the biological function of miR-150. Finally, miR-150 overexpression and RUNX2 knockdown enhanced caspase-3 cleavage. Taken together, the present study established a novel molecular mechanism, in that miR-150 plays tumor suppressor and chemoprotective roles by targeting RUNX2 in OS, indicating that miR-150 may be a potential therapeutic target for OS therapy in the future.

Covalently polysaccharide-based alginate/chitosan hydrogel embedded alginate microspheres for BSA encapsulation and soft tissue engineering.

Hydrogels based scaffolds are very promising materials for a wide range of medical applications including tissue engineering and drug delivery. This study reports a covalently cross-linked composite hydrogel embedded with microspheres basing natural polysaccharides as a protein delivery system for soft tissue engineering. This biodegradable composite hydrogel derived from water-soluble chitosan and alginate derivatives upon mixing, without addition of chemical cross-linking agents. The gelation is attributed to the Schiff-base reaction between amino and aldehyde groups of N-succinyl chitosan (N-Chi) and oxidized alginate (OAlg), respectively. Meanwhile, gel-like microspheres were prepared with a diameter of 2-10 µm by conjugating sodium alginate with Ca2+ in an aqueous emulsion via the emulsion cross-linking technique. Bull Serum Albumin (BSA) was encapsulated into alginate gel microspheres and subsequently incorporated into OAlg/N-Chi hydrogels to produce a composite scaffold. In the current work, gelation rate, morphology, mechanical properties, swelling ratio, in vitro degradation and BSA release of the composite scaffolds were examined. The results show that mechanical and stable properties of gel scaffolds can be significantly improved by embedding alginate microspheres. The alginate microspheres can serve as a filler to toughen the soft OAlg/N-Chi hydrogels. Compressive modulus of composite gel scaffolds containing 0.5 mL volume of microspheres was 57.3 KPa, which was higher than the control hydrogel without microspheres. Moreover, the controlled release of BSA encapsulated within this composite hydrogels showed significantly lower rate when compared with control hydrogel or microspheres alone. These characteristics provide a potential opportunity to use this injectable composite gel scaffold in protein delivery and soft tissue engineering applications.

Carbon nanotubes as electrophysiological building blocks for a bioactive cell scaffold through biological assembly to induce osteogenesis.

Bio-functional cell scaffolds have great potential in the field of tissue regenerative medicine. In this work, a carbon nanotube (CNT) gel scaffold via specific pairing of functionalized nucleobases was developed for specifically targeted drug delivery and in vitro osteogenesis. The CNT gel scaffold with nano-fibrous architectures was established by Watson-Crick base pairing between thymine and adenine of low molecular weight heparin, respectively. As scaffold precursors, adenine and thymine functionalized heparin derivatives could additionally bind cell growth factors by the affinity interaction. The resulting nano-fibrous gel scaffolds showed excellent mechanical integrity and advanced electro-physiological functions. Potential application of the electrophysiological CNT gel scaffold in bone tissue engineering was confirmed by encapsulation of human adipose-derived stem cells (ASCs). Our results indicate that the electrically conductive networks formed by CNTs within the nano-fibrous framework are the key characteristics of cell scaffolds leading to improved ASC organization and differentiation by an extra electrical stimulus (ES). Specifically, ASCs cultured in bio-electrical gel scaffolds showed ∼4 times higher spontaneous osteogenesis in combination with bone morphogenetic protein 2 (BMP-2), compared to those cultured on pristine hydrogels. This electrophysiological CNT gel scaffold containing BMP-2 exhibited beneficial effects on ASC activity and osteogenetic differentiation, which suggested a promising future for local treatment of bone regeneration.

Injectable polysaccharide hydrogel embedded with hydroxyapatite and calcium carbonate for drug delivery and bone tissue engineering.

To meet the progressive requirements for bone regeneration purpose, injectable hydrogels have attracted increasing attention in tissue regeneration and local drug delivery applications. In this study, we report a facile method to prepare injectable and degradable polysaccharide-based hydrogels doubly integrated with hydroxyapatite (HAp) nanoparticles and calcium carbonate microspheres (CMs) under physiological condition. The mechanism of cross-linking is attributed to the Schiff-base reaction between amino and aldehyde groups of carboxymethyl chitosan (CMCS) and oxidized alginate (OAlg), respectively. Synchronously, tetracycline hydrochloride (TH) loaded CMs were fabricated by the precipitation reaction with an average diameter of 6.62 µm. To enhance bioactive and mechanical properties, nano-HAp and CMs containing TH were encapsulated into the polysaccharide-based hydrogel to form injectable gel scaffolds for imitation of bone niche. The gelation time, morphology, mechanical properties, swelling ratio and in vitro degradation of the gel scaffolds could be controlled by varying HAp and CMs contents. Moreover, the composite gel scaffolds had good sustained drug release and antibacterial properties, as confirmed by drugs release calculation and antibacterial evaluation. In addition, the gel scaffolds were found to be self-healing due to dynamic equilibrium of the Schiff-base linkages. These results suggested that the prepared composite gel scaffolds hold great potential for drug delivery and regeneration of irregular bone defects.

Dynamical release nanospheres containing cell growth factor from biopolymer hydrogel via reversible covalent conjugation.

For practical adipose regeneration, the challenge is to dynamically deliver the key adipogenic insulin-like growth factors in hydrogels to induce adipogenesis. In order to achieve dynamic release, smart hydrogels to sense the change in the blood glucose concentration is required when glucose concentration increases. In this study, a heparin-based hydrogel has been developed for use in dynamic delivery of heparin nanospheres containing insulin-like growth factor. The gel scaffold was facilely prepared in physiological conditions by the formation of boronate-maltose ester cross-links between boronate and maltose groups of heparin derivatives. Due to its intrinsic glucose-sensitivity, the exposure of gel scaffold to glucose induces maltose functionalized nanospheres dissociation off hydrogel network and thereby could dynamically move into the microenvironment. The potential of the hydrogel as a cell scaffold was demonstrated by encapsulation of human adipose-derived stem cells (ASCs) within the gel matrix in vitro. Cell culture showed that this dynamic hydrogel could support survival and proliferation of ASCs. This biocompatible coupling chemistry has the advantage that it introduces no potentially cytotoxic groups into injectable gel scaffolds formed and can create a more biomimetic microenvironment for drug and cell delivery, rendering them more suitable for potential in vivo biomedical applications. All these results indicate that this biocompatible gel scaffold can render the formulation of a therapeutically effective platform for diabetes treatment and adipose regeneration.

Chitosan membrane dressings toughened by glycerol to load antibacterial drugs for wound healing.

Transparent and flexible chitosan-based membranes containing antibacterial drugs were prepared through a casting/solvent evaporation method from suspension of chitosan floccule. To enhance mechanical properties, glycerin was incorporated into the floccule suspension to conjugate chitosan as a plasticizer. The mechanism of membrane formation is attributed to inter- and intro-hydrogen bonding between chitosan and glycerol molecules. The results showed that incorporation of glycerol has a significant influence on the properties of the chitosan membranes. With the increase of glycerol content, the tensile strength, swelling rate, water vapor permeability and wettability of membranes were significantly improved. In vitro enzymatic degradation revealed that the chitosan membrane had long-term stability regardless of the glycerol content. To enhance antibacterial properties, tetracycline hydrochloride (TH) and silver sulfadiazine (AgSD), representing the water-soluble and water-insoluble drug models, were integrated into the membranes, respectively. The results of controlled-release efficacy and inhibition zone indicate that the glycerol toughened chitosan membranes containing drugs have a promising future in treatment of bacterial infection as wound dressing.

Covalent and injectable chitosan-chondroitin sulfate hydrogels embedded with chitosan microspheres for drug delivery and tissue engineering.

Injectable hydrogels and microspheres derived from natural polysaccharides have been extensively investigated as drug delivery systems and cell scaffolds. In this study, we report a preparation of covalent hydrogels basing polysaccharides via the Schiff' base reaction. Water soluble carboxymethyl chitosan (CMC) and oxidized chondroitin sulfate (OCS) were prepared for cross-linking of hydrogels. The mechanism of cross-linking is attributed to the Schiff' base reaction between amino and aldehyde groups of polysaccharides. Furthermore, bovine serum albumin (BSA) loaded chitosan-based microspheres (CMs) with a diameter of 3.8-61.6µm were fabricated by an emulsion cross-linking method, followed by embedding into CMC-OCS hydrogels to produce a composite CMs/gel scaffold. In the current work, gelation rate, morphology, mechanical properties, swelling ratio, in vitro degradation and BSA release of the CMs/gel scaffolds were examined. The results show that mechanical and bioactive properties of gel scaffolds can be significantly improved by embedding CMs. The solid CMs can serve as a filler to toughen the soft CMC-OCS hydrogels. Compressive modulus of composite gel scaffolds containing 20mg/ml of microspheres was 13KPa, which was higher than the control hydrogel without CMs. Cumulative release of BSA during 2weeks from CMs embedded hydrogel was 30%, which was significantly lower than those of CMs and hydrogels. Moreover, the composite CMs/gel scaffolds exhibited lower swelling ratio and slower degradation rate than the control hydrogel without CMs. The potential of the composite hydrogel as an injectable scaffold was demonstrated by encapsulation of bovine articular chondrocytes in vitro. These results demonstrate the potential of CMs embedded CMC-OCS hydrogels as an injectable drug and cell delivery system in cartilage tissue engineering.

Covalently antibacterial alginate-chitosan hydrogel dressing integrated gelatin microspheres containing tetracycline hydrochloride for wound healing.

An antibacterial and biodegradable composite hydrogel dressing integrated with microspheres is developed for drug delivery and wound healing. The mechanism of gelation is attributed to the Schiff-base reaction between aldehyde and amino groups of oxidized alginate (OAlg) and carboxymethyl chitosan (CMCS). To enhance antibacterial and mechanical properties, tetracycline hydrochloride (TH) loaded gelatin microspheres (GMs) were fabricated by an emulsion cross-linking method, followed by integrating into the OAlg-CMCS hydrogel to produce a composite gel dressing. In vitro gelation time, swelling, degradation, compressive modulus and rheological properties of the gel dressing were investigated as the function of microsphere ratios. With increasing ratios of microspheres from 10 to 40mg/mL, the composite dressing manifested shorter gelation time and lower swelling ratios, as well as higher mechanical strength. Comparing to other formulations, the gel dressing with 30mg/mL microspheres showed more suitable stabilities and mechanical properties for wound healing. Also, in vitro drug release results showed that the loaded TH could be sustained release from the composite gel dressing by contrast with pure hydrogels and microspheres. Furthermore, powerful bacteria growth inhibition effects against Escherichia coli and Staphylococcus aureus suggested that the composite gel dressing, especially the one with 30mg/mL GMs containing TH, has a promising future in treatment of bacterial infection.

Injectable alginate/hydroxyapatite gel scaffold combined with gelatin microspheres for drug delivery and bone tissue engineering.

Injectable and biodegradable alginate-based composite gel scaffolds doubly integrated with hydroxyapatite (HAp) and gelatin microspheres (GMs) were cross-linked via in situ release of calcium cations. As triggers of calcium cations, CaCO3 and glucono-D-lactone (GDL) were fixed as a mass ratio of 1:1 to control pH value ranging from 6.8 to 7.2 during gelation. Synchronously, tetracycline hydrochloride (TH) was encapsulated into GMs to enhance bioactivity of composite gel scaffolds. The effects of HAp and GMs on characteristics of gel scaffolds, including pH value, gelation time, mechanical properties, swelling ratio, degradation behavior and drug release, were investigated. The results showed that HAp and GMs successfully improved mechanical properties of gel scaffolds at strain from 0.1 to 0.5, which stabilized the gel network and decreased weight loss, as well as swelling ratio and gelation time. TH could be released from this composite gel scaffold into the local microenvironment in a controlled fashion by the organic/inorganic hybrid of hydrogel network. Our results demonstrate that the HAp and GMs doubly integrated alginate-based gel scaffolds, especially the one with 6% (w/v) HAp and 5% (w/v) GMs, have suitable physical performance and bioactive properties, thus provide a potential opportunity to be used for bone tissue engineering. The potential application of this gel scaffold in bone tissue engineering was confirmed by encapsulation behavior of osteoblasts. In combination with TH, the gel scaffold exhibited beneficial effects on osteoblast activity, which suggested a promising future for local treatment of pathologies involving bone loss.

Magnetic hyaluronic acid nanospheres via aqueous Diels-Alder chemistry to deliver dexamethasone for adipose tissue engineering.

Biopolymer-based nanospheres have great potential in the field of drug delivery and tissue regenerative medicine. In this work, we present a flexible way to conjugate a magnetic hyaluronic acid (HA) nanosphere system that are capable of vectoring delivery of adipogenic factor, e.g. dexamethasone, for adipose tissue engineering. Conjugation of nanospheres was established by aqueous Diels-Alder chemistry between furan and maleimide of HA derivatives. Simultaneously, a furan functionalized dexamethasone peptide, GQPGK, was synthesized and covalently immobilized into the nanospheres. The magnetic HA nanospheres were fabricated by encapsulating super-paramagnetic iron oxide nanoparticles, which exhibited quick magnetic sensitivity. The aqueous Diels-Alder chemistry made nanospheres high binding efficiency of dexamethasone, and the vectoring delivery of dexamethasone could be easily controlled by a external magnetic field. The potential application of the magnetic HA nanospheres on vectoring delivery of adipogenic factor was confirmed by co-culture of human adipose-derived stem cells (ASCs). In vitro cytotoxicity tests demonstrated that incorporation of dexamethasone into magnetic HA nanospheres showed high efficiency to promote ASCs viabilities, in particular under a magnetic field, which suggested a promising future for adipose regeneration applications.