Effects of general and spinal anesthesia on postoperative rehabilitation in older adults after lower limb surgery: a retrospective cohort study.

Objective: To investigate the effects of perioperative general anesthesia (GA) and spinal anesthesia (SA) on postoperative rehabilitation in elderly patients with lower limb surgery. Methods: This retrospective propensity score-matched cohort study included patients aged 65 years or older who underwent lower limb surgery between January 1, 2020, and May 31, 2023. The GA and SA were selected at the request of the orthopedic surgeon, patient, and their family members. The main outcomes included the incidence of the patient's inability to self-care at discharge, postoperative complications including pulmonary infection, thrombus of lower extremity veins, infection of incisional wound and delirium, length of hospital stay, and incidence of severe pain in the first 2 days postoperatively. Results: In total, 697 patients met the inclusion criteria, and 456 were included in the final analysis after propensity score matching. In the GA and SA groups, 27 (11.84%) and 26 (11.40%) patients, respectively, could not care for themselves at discharge. The incidence rates did not differ between the groups (p = 0.884). In contrast, the incidence of postoperative complications (GA: 10.53% and SA: 4.39%; p = 0.013) and the length of hospital stay (GA: 16.92 ± 10.65 days and SA: 12.75 ± 9.15 days; p < 0.001) significantly differed between the groups. Conclusion: The choice of anesthesia is independent of the loss of postoperative self-care ability in older patients (>65 years) and is not a key factor affecting postoperative rehabilitation after lower limb surgery. However, compared with GA, SA reduces the incidence of postoperative complications and a prolonged hospital stay. Thus, SA as the primary anesthetic method is a protective factor against a prolonged hospital stay.

[Effect of sodium alginate-g-deferoxamine/chitosan microspheres on osteogenic differentiation of rat bone mesenchymal stem cells].

PURPOSE: To explore the effect of sodium alginate-g-deferoxamine/chitosan (SA-g-DFO/CS) microspheres on proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs). METHODS: A kind of SA-g-DFO/CS microsphere was developed through electrostatic interaction between porous chitosan microspheres and sodium alginate chemically grafted on the surface of DFO. Its morphology, porosity rate, pore size and sustained release of DFO in vitro were examined. Rat BMSCs were isolated and co-cultured with microspheres in osteogenic differentiation medium. MTT assay was used to study the influence of cell proliferation, and Calcein-AM/PI staining was used to observe the cell viability. Alkaline phosphatase (ALP) activity assay was conducted. PCR was used to detect the expression of genes related to angiogenesis and osteogenesis. Statistical analysis was performed using SPSS 22.0 software package. RESULTS: The SA-g-DFO/CS porous microspheres were successfully prepared with a sustained re6lease of DFO. Compared with SA/CS microspheres, the SA-g-DFO/CS microspheres were conducive to cell proliferation and differentiation, with the increases in expression level of ALP, related angiogenesis genes HIF-1α, VEGF and osteogenesis genes COLI, OCN. CONCLUSIONS: The SA-g-DFO/CS porous microspheres can provide a new choice for the development of alveolar bone regeneration.

Injectable, self-healing poly(amino acid)-hydrogel based on phenylboronate ester bond for osteochondral tissue engineering.

A new generation of osteochondral integrated scaffolds is needed for articular osteochondral regeneration, which can not only facilitate the accurate construction of osteochondral scaffolds in a minimally invasive manner but also firmly combine the subchondral bone layer and cartilage layer. Herein, an osteochondral integrated hydrogel scaffold was constructed by the poly(L-glutamic acid) (PLGA) based self-healing hydrogels with phenylboronate ester (PBE) as the dynamic cross-linking. The bone layer self-healing hydrogel (hydrogel O-S) was prepared by physically blending nanohydroxyapatite into the self-healing hydrogel PLGA-PBE-S, which was fabricated by 3-aminophenylboronic acid/glycidyl methacrylate-modified PLGA (PLGA-GMA-PBA) and 3-amino-1,2-propanediol/N-(2-aminoethyl) acrylamide-modified PLGA (PLGA-ADE-AP). The cartilage layer self-healing hydrogel (hydrogel C-S) was prepared by PLGA-GMA-APBA and glucosamine- modified PLGA-ADE-AP (PLGA-ADE-AP-G). Excellent injectability and self-healing profiles of hydrogel O-S and C-S were observed, the self-healing efficiencies were 97.02% ± 1.06% and 99.06% ± 0.57%, respectively. Based on the injectability and spontaneous healing on the interfaces of hydrogel O-S and C-S, the osteochondral hydrogel (hydrogel OC) was conveniently constructed in a minimally invasive manner. In addition,in situphotocrosslinking was used to enhance the mechanical strength and stability of the osteochondral hydrogel. The osteochondral hydrogels exhibited good biodegradability and biocompatibility. The osteogenic differentiation genes BMP-2, ALPL, BGLAP and COL I of adipose-derived stem cells (ASCs) in the bone layer of the osteochondral hydrogel were significantly expressed, and the chondrogenic differentiation genes SOX9, aggrecan and COL II of ASCs in the cartilage layer of the osteochondral hydrogel were obviously upregulated after 14 d of induction. The osteochondral hydrogels could effectively promote repair of osteochondral defects after 3 months post-surgery.

Building a Poly(amino acid)/Chitosan-Based Self-Healing Hydrogel via Host-Guest Interaction for Cartilage Regeneration.

Cartilage injury is a very common joint disease, and cartilage repair is a great challenge in clinical treatment due to the specific structure of cartilage tissue and its microenvironment in vivo. The injectable self-healing hydrogel is a very promising candidate as a cartilage repair material because of its special network structure, high water retention and self-healing properties. In this work, a self-healing hydrogel cross-linked by host-guest interaction between cyclodextrin and cholic acid was developed. The host material was composed of ß-cyclodextrin and 2-hydroxyethyl methacrylate-modified poly(l-glutamic acid) (P(LGA-co-GM-co-GC)), while the guest material was chitosan modified by cholic acid, glycidyl methacrylate, and (2,3-epoxypropyl)trimethylammonium chloride (EPTAC) (QCSG-CA). The host-guest interaction self-healing hydrogels, named as HG hydrogels (HG gel), exhibited excellent injectability and self-healable property, and the self-healing efficiency was greater than 90%. Furthermore, in order to enhance the mechanical properties and slow down the degradation of the HG gel in vivo, the second network was constructed by photo-cross-linking in situ. Biocompatibility tests showed that the enhanced multi-interaction hydrogel (MI gel) was extremely suitable for cartilage tissue engineering both in vitro and in vivo. In addition, the adipose derived stem cells (ASCs) in MI gel were able to differentiate cartilage effectively in vitro in the presence of inducing agents. Subsequently, the MI gel without ASCs was transplanted into rat cartilage defects in vivo for the regeneration of cartilage. After 3 months postimplantation, new cartilage tissue was successfully regenerated in a rat cartilage defect. All results indicated that the injectable self-healing host-guest hydrogels have important potential applications in cartilage injury repair.

Injectable macro-porous chitosan/polyethylene glycol-silicotungstic acid double-network hydrogels based on "smashed gels recombination" strategy for cartilage tissue engineering.

The lack of interconnected macro-porous structure of most injectable hydrogels lead to poor cell and tissue infiltration. Herein, we present the fabrication of injectable macro-porous hydrogels based on "smashed gels recombination" strategy. Chitosan/polyethylene glycol-silicotungstic acid (CS/PEG-SiW) double-network hydrogels were prepared via dual dynamic interactions. The bulk CS/PEG-SiW hydrogels were then smashed into micro-hydrogels with average sizes ranging from 47.6 to 63.8 µm by mechanical fragmentation. The CS/PEG-SiW micro-hydrogels could be continuously injected and rapidly recombined into a stable porous hydrogel based on the dual dynamic interactions between micro-hydrogels. The average pore size of the recombined porous CS/PEG-SiW hydrogels ranged from 52 to 184 µm. The storage modulus, compress modulus and maximum compressive strain of the recombined porous CS/PEG-SiW1.0 hydrogels reached about 47.2 %, 28.2 % and 127.6 % of the values for their corresponding bulk hydrogels, respectively. The recombined porous hydrogels were cytocompatible and could effectively support proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In a rat cartilage defect model, recombined porous CS/PEG-SiW hydrogels could promote cartilage regeneration. Hematoxylin and eosin (H&E), Safranin-O/Fast green and immunohistochemical staining confirmed the accumulation of glycosaminoglycans (GAG) and type II collagen (Col II) in regenerated cartilage.

Hexokinase 2 Is a Pivot for Lovastatin-induced Glycolysis-to-Autophagy Reprogramming in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited therapeutic options available. We have recently demonstrated that lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, suppresses TNBC cell proliferation and stemness properties in vitro and in vivo. However, the mechanisms through which lovastatin inhibits TNBC cells are not fully understood. Here, we used 1H NMR-based metabolomic profiling to investigate lovastatin-induced metabolic changes in TNBC cell line MDA-MB-231. Among the 46 metabolites identified, lactate demonstrated the highest variable importance in projection (VIP) score. Glycolysis stress test revealed that lovastatin significantly decreased the extracellular acidification rate (ECAR) in MDA-MB-231 cells. Furthermore, lovastatin treatment down-regulated the levels of glycolysis-related proteins including GLUT1, PFK1, and PKM2 in MDA-MB-231 but not non-TNBC MDA-MB-453 cells. In addition, lovastatin induced autophagy as evidenced by increased LC3 puncta formation and LC3-II/I ratio, increased AMPK phosphorylation, and decreased Akt phosphorylation. We also revealed the interaction between the glycolytic enzyme hexokinase 2 (HK2) and the mitochondrial membrane protein voltage-dependent anion channel 1 (VDAC1), an important regulator of autophagy. Further bioinformatics analysis revealed that VDAC1 was expressed at a higher level in breast cancer than normal tissues and higher level of VDAC1 predicted poorer survival outcomes in breast cancer patients. The present study suggests that lovastatin might exert anti-tumor activity by reprogramming glycolysis toward autophagy in TNBC cells through HK2-VDAC1 interaction.

Preparation of Assemblable Chondral and Subchondral Bone Microtissues for Osteochondral Tissue Engineering.

Microtissues exhibit great advantages in injecting with minimum invasiveness, mimicking natural tissues, and promoting tissue regeneration. However, very few studies have focused on the construction of osteochondral microtissues that could simultaneously support hyaline-like cartilage and bone tissue regeneration. In this study, chondral microtissues that could favor the formation of hyaline-like cartilages and subchondral bone microtissues that could repair subchondral defects to support the neo-generated cartilages were successfully constructed for osteochondral tissue engineering. For chondral repair, the developed chondral microgels with high porosity and hydrophilicity could make cells spherical, favor the formation of cell aggregates, and show an excellent differentiation effect toward hyaline-like cartilage, thus contributing to the production of chondral microtissues. For subchondral bone repair, the fabricated subchondral microgels realize cell adhesion and proliferation and support the osteogenic differentiation of stem cells, thus favoring the formation of subchondral bone microtissues. The injectable chondral and subchondral bone microtissues could be stably assembled by Michael addition reaction between sulfhydryl groups of microtissues and double bonds of hydrophilic macromolecular cross-linker. At 12 weeks postimplantation, osteochondral microtissues could support the reconstruction of osteochondral-like tissues. The present study provides new insight into the microtissues for repair of osteochondral tissues.

Nucleolar and Coiled-Body Phosphoprotein 1 Is Associated With Stemness and Represents a Potential Therapeutic Target in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and lacks approved specific targeted therapies. One of the major reasons why TNBC is difficult to treat is the high proportion of cancer stem cells within the tumor tissue. Nucleolus is the location of ribosome biogenesis which is frequently overactivated in cancer cells and overactivation of ribosome biogenesis frequently drives the malignant transformation of cancer. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) is a nucleolar protein responsible for nucleolus organization and rRNA synthesis and plays an important role in ribosome biogenesis. However, the correlation of NOLC1 expression with patient prognosis and its value as a therapeutic target have not been evaluated in TNBC. In the current study, based on bioinformatics analysis of the online databases, we found that the expression of NOLC1 was higher in breast cancer tissues than normal tissues, and NOLC1 was expressed at a higher level in TNBC than other subtypes of breast cancer. GSEA analysis revealed that stemness-related pathways were significantly enriched in breast cancer with high NOLC1 gene expression. Further analyses using gene expression profiling interactive analysis 2 (GEPIA2), tumor immune estimation resource (TIMER) and search tool for retrieval of interacting genes/proteins (STRING) demonstrated that NOLC1 was significantly associated with stemness in both all breast cancer and basal-like breast cancer/TNBC patients at both gene and protein levels. Knockdown of NOLC1 by siRNA decreased the protein level of the key stemness regulators MYC and ALDH and inhibited the sphere-forming capacity in TNBC cell line MDA-MB-231. Univariate and multivariate Cox regression analyses demonstrated that NOLC1 was an independent risk factor for overall survival in breast cancer. PrognoScan and Kaplan-Meier plotter analyses revealed that high expression of NOLC1 was associated with poor prognosis in both all breast cancer and TNBC patients. Further immunohistochemical analysis of breast cancer patient samples revealed that TNBC cells had a lower level of NOLC1 in the nucleus compared with non-TNBC cells. These findings suggest that NOLC1 is closely associated with the stemness properties of TNBC and represents a potential therapeutic target for TNBC.

Krüppel-like factor 5-induced overexpression of long non-coding RNA DANCR promotes the progression of cervical cancer via repressing microRNA-145-3p to target ZEB1.

Long non-coding RNA (lncRNA) differentiation antagonizing non-protein coding RNA (DANCR) participates in the development of diverse cancers. Nevertheless, the impact of DANCR on cervical cancer (CC) remains largely unknown. This study aims to explore the effects of DANCR sponging microRNA-145-3p (miR-145-3p) on CC. Expression of KLF5, DANCR, miR-145-3p, and zinc finger E-box binding homeobox 1 (ZEB1) in CC and adjacent normal tissues was determined. Human CC cell lines were, respectively, treated with silenced DANCR or miR145-3p mimic/inhibitor. Then, the viability, migration, invasion, and apoptosis of CC cells were measured. The cell growth in vivo was observed as well. Chromatin immunoprecipitation assay was performed to analyze the binding of KLF5 and DANCR promoter. Interaction among DANCR, miR-145-3p, and ZEB1 was assessed. KLF5, DANCR, and ZEB1 were upregulated but miR-145-3p was downregulated in CC tissues. KLF5 activated DANCR expression and the high DANCR expression was related to tumor staging, infiltrating muscle depth and lymphatic metastasis of CC patients. Reduced DANCR or elevated miR-145-3p repressed malignant behaviors of CC cells. The tumor diameter and weight were also repressed by DANCR silencing or miR-145-3p elevation. The effect of DANCR knockdown on CC cells could be reversed by miR-145-3p inhibitor. MiR-145-3p was targeted by DANCR and ZEB1 was targeted by miR-145-3p. KLF5-induced overexpression of DANCR promotes CC progression via suppressing miR-145-3p to target ZEB1. This study may provide potential targets for CC treatment.

Mussel-Inspired Bisphosphonated Injectable Nanocomposite Hydrogels with Adhesive, Self-Healing, and Osteogenic Properties for Bone Regeneration.

Injectable hydrogels have received much attention because of the advantages of simulation of the natural extracellular matrix, microinvasive implantation, and filling and repairing of complex shape defects. Yet, for bone repair, the current injectable hydrogels have shown significant limitations such as the lack of tissue adhesion, deficiency of self-healing ability, and absence of osteogenic activity. Herein, a strategy to construct mussel-inspired bisphosphonated injectable nanocomposite hydrogels with adhesive, self-healing, and osteogenic properties is developed. The nano-hydroxyapatite/poly(l-glutamic acid)-dextran (nHA/PLGA-Dex) dually cross-linked (DC) injectable hydrogels are fabricated via Schiff base cross-linking and noncovalent nHA-BP chelation. The chelation between bisphosphonate ligands (alendronate sodium, BP) and nHA favors the uniform dispersion of the latter. Moreover, multiple adhesion ligands based on catechol motifs, BP, and aldehyde groups endow the hydrogels with good tissue adhesion. The hydrogels possess excellent biocompatibility and the introduction of BP and nHA both can effectively promote viability, proliferation, migration, and osteogenesis differentiation of MC3T3-E1 cells. The incorporation of BP groups and HA nanoparticles could also facilitate the angiogenic property of endothelial cells. The nHA/PLGA-Dex DC hydrogels exhibited considerable biocompatibility despite the presence of a certain degree of inflammatory response in the early stage. The successful healing of a rat cranial defect further proves the bone regeneration ability of nHA/PLGA-Dex DC injectable hydrogels. The developed tissue adhesive osteogenic injectable nHA/PLGA-Dex hydrogels show significant potential for bone regeneration application.

Lovastatin Inhibits EMT and Metastasis of Triple-Negative Breast Cancer Stem Cells Through Dysregulation of Cytoskeleton-Associated Proteins.

Triple-negative breast cancer (TNBC) is more aggressive and has poorer prognosis compared to other subtypes of breast cancer. Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells transform into mesenchymal-like cells capable of migration, invasion, and metastasis. Recently, we have demonstrated that lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor and a lipid-lowering drug, could inhibit stemness properties of cancer stem cells (CSCs) derived from TNBC cell in vitro and in vivo. This study is aimed at investigating whether lovastatin inhibits TNBC CSCs by inhibiting EMT and suppressing metastasis and the mechanism involved. In the present study, we found that lovastatin dysregulated lysine succinylation of cytoskeleton-associated proteins in CSCs derived from TNBC MDA-MB-231 cell. Lovastatin inhibited EMT as demonstrated by down-regulation of the protein levels of Vimentin and Twist in MDA-MB-231 CSCs in vitro and vivo and by reversal of TGF-ß1-induced morphological change in MCF10A cells. Lovastatin also inhibited the migration of MDA-MB-231 CSCs. The disruption of cytoskeleton in TNBC CSCs by lovastatin was demonstrated by the reduction of the number of pseudopodia and the relocation of F-actin cytoskeleton. Combination of lovastatin with doxorubicin synergistically inhibited liver metastasis of MDA-MB-231 CSCs. Bioinformatics analysis revealed that higher expression levels of cytoskeleton-associated genes were characteristic of TNBC and predicted survival outcomes in breast cancer patients. These data suggested that lovastatin could inhibit the EMT and metastasis of TNBC CSCs in vitro and in vivo through dysregulation of cytoskeleton-associated proteins.

Artemisinin Derivatives Inhibit Non-small Cell Lung Cancer Cells Through Induction of ROS-dependent Apoptosis/Ferroptosis.

Non-small cell lung cancer (NSCLC) is one of the major cancer-related causes of morbidity and mortality worldwide. Despite the progress in lung cancer treatment, there is still an urgent need to discover novel therapeutic agents for NSCLC. Natural products represent a rich source of bioactive compounds. Through a natural compound library screening assay, we found that a group of anti-insect drugs had significant inhibitory effect on the proliferation of NSCLC cells. Among the anti-insect drugs, two derivatives of artemisinin, i.e., artesunate (ART) and dihydroartemisinin (DHA), a group of well-known anti-malarial drugs, have been shown to possess selective anti-cancer properties. Mechanistically, we found that ART and DHA induced apoptosis of A549 cells as evidenced by decreased protein level of VDAC and increased caspase 3 cleavage. Furthermore, cystine/glutamate transporter (xCT), a core negative regulator of ferroptosis, was downregulated by ART and DHA. The mRNA level of transferrin receptor (TFRC), a positive regulator of ferroptosis, was upregulated by ART and DHA. ART/DHA-induced apoptosis and ferroptosis in NSCLC cells were partly reversed by N-Acetyl-L-cysteine (NAC), a ROS scavenger, and ferrostatin-1, a ferroptosis inhibitor, respectively. These results suggest that artemisinin derivatives have anti-NSCLC activity through induction of ROS-dependent apoptosis/ferroptosis. Our findings provide the experimental basis for the potential application of artemisinin derivatives as a class of novel therapeutic drugs for NSCLC.

Designer Hydrogel with Intelligently Switchable Stem-Cell Contact for Incubating Cartilaginous Microtissues.

Stem-cell-derived organoid can resemble in vivo tissue counterpart and mimic at least one function of tissue or organ, possessing great potential for biomedical application. The present study develops a hydrogel with cell-responsive switch to guide spontaneous and sequential proliferation and aggregation of adipose-derived stem cells (ASCs) without inputting artificial stimulus for in vitro constructing cartilaginous microtissues with enhanced retention of cell-matrix and cell-cell interactions. Polylactic acid (PLA) rods are surface-aminolyzed by cystamine, followed by being involved in the amidation of poly(( l-glutamic acid) and adipic acid dihydrazide (ADH) to form a hydrogel. Along with tubular pore formation in hydrogel after dissolution of PLA rods, aminolyzed PLA molecules with disulfide bonds on rod surfaces are covalently transferred to the tubular pore surfaces of poly(l-glutamic acid)/ADH hydrogel. Because PLA attaches cells, while poly(l-glutamic acid)/ADH hydrogel repels cells, ASCs are found to adhere and proliferate on the tubular pore surfaces of hydrogel first and then cleave disulfide bonds by secreting molecules containing thiol, thus inducing desorption of PLA molecules and leading to their spontaneous detachment and aggregation. Associated with chondrogenic induction by TGF-ß1 and IGF-1 in vitro for 28 days, the hydrogel as an all-in-one incubator produces well-engineered columnar cartilage microtissues from ASCs, with the glycosaminoglycans (GAGs) and collagen type II (COL II) deposition achieving 64 and 69% of those in chondrocytes pellet, respectively. The cartilage microtissues further matured in vivo for 8 weeks to exhibit extremely similar histological features and biomechanical performance to native hyaline cartilage. The GAGs and COL II content, as well as compressive modulus of the matured tissue show no significant difference with native cartilage. The designer hydrogel may hold a promise for long-term culture of other types of stem cells and organoids.

In Situ Precipitation of Cluster and Acicular Hydroxyapatite onto Porous Poly(γ-benzyl-l-glutamate) Microcarriers for Bone Tissue Engineering.

Bone tissue engineering scaffold based on microcarriers provides an effective approach for the repair of irregular bone defects. The implantation of microcarriers by injection can reduce surgical trauma and fill various irregular shaped bone defects. Microcarriers with porous structure and osteogenic properties have shown great potential in promoting the repair of bone defects. In this study, two kinds of hydroxyapatite/poly-(γ-benzyl-l-glutamate) (HA/PBLG) microcarriers were constructed by emulsion/in situ precipitation method and their structures and properties were studied. First, PBLG porous microcarriers were prepared by an emulsion method. Surface carboxylation of PBLG microcarriers was performed to promote the deposition of HA on PBLG microcarriers. Next, the modified porous PBLG microcarriers were used as the matrix, combined with the in situ precipitation method; the cluster HA and acicular HA were precipitated onto the surface of porous microcarriers in the presence of ammonia water and tri(hydroxymethyl)aminomethane (Tris) solution, respectively. The micromorphology, composition, and element distribution of the two kinds of microcarriers were characterized by TEM, SEM, and AFM. Adipose stem cells (ADSCs) were cultured on the cluster HA/PBLG and acicular HA/PBLG microcarriers, respectively. ADSCs could grow and proliferate normally on both kinds of microcarriers wherein the acicular HA/PBLG microcarriers were more favorable for early cell adhesion and showed a beneficial effect on mineralization and osteogenic differentiation of ADSCs. Successful healing of a rabbit femur defect verified the bone regeneration ability of acicular HA/PBLG microcarriers.

All-in-One Hydrogel Realizing Adipose-Derived Stem Cell Spheroid Production and In Vivo Injection via "Gel-Sol" Transition for Angiogenesis in Hind Limb Ischemia.

Adipose-derived stem cell (ASC) spheroids exhibit enhanced angiogenic efficacy toward ischemia treatment. Thus, it is necessary to develop an all-in-one platform that enables efficient spheroid production, collection, and injectable implantation in vivo. The present study fabricated a poly(l-glutamic acid) (PLGA)-based porous hydrogel that can not only produce ASC spheroids but also conveniently collect spheroids for in vivo implantation via minimally invasive injection to treat hind limb ischemia. PLGA was cross-linked with cystamine (Cys), which contains disulfide bonds, to form a porous hydrogel that could realize "gel-sol" transition by the reduction effect of glutathione (GSH). For one thing, it was found that the introduction of the disulfide bond in the PLGA hydrogel promoted cellular adhesion via combining fibronectin, preventing the formation of spheroids, while the introduction of polyethylene glycol monomethyl ether (mPEG) could disturb the effect of the disulfide bond on cellular adhesion, supporting spheroid formation inside the porous hydrogel. For another, the porous hydrogel transferred into a syringe could turn into liquid polymer solution within about 40 min for collection of the produced spheroids and in vivo injection. In addition, because of the lubrication of polymer solution, the spheroids were protected during the injection of the spheroids/polymer suspensoid through a 25G syringe needle, avoiding damages from shearing. After the in vivo injection, the enhanced paracrine secretion of ASC spheroids resulted in promoted angiogenesis and muscle regeneration, exhibiting obvious therapeutic effect on limb ischemia in mice after 21 days. At the same time, PLGA-based material exhibited well-performed biocompatibility in vivo.

Thermoresponsive Chitosan/DOPA-Based Hydrogel as an Injectable Therapy Approach for Tissue-Adhesion and Hemostasis.

Chitosan (CS) hydrogels are widely used in wound hemostatic agents due to their superior biocompatibility, biodegradability, and hemostatic effect. However, most of them fail to achieve great hemostatic effect because of poor adhesion to bleeding tissues. Also, the conventional implantation surgery of hemostatic hydrogels to internal bleeding wounds may cause secondary trauma to the human body. In this work, catechol-hydroxybutyl chitosan (HBCS-C) has been designed and prepared by grafting hydroxybutyl groups and catechol groups to the CS backbones. The multifunctional HBCS-C hydrogels are fabricated with the properties of thermosensitivity, injectability, tissue-adhesion, biodegradation, biocompatibility, and wound hemostasis. They exhibit excellent liquid-gel transition at different temperatures, through the changes of hydrophilic-hydrophobic interaction and hydrogen bonds generating from hydroxybutyl groups. By the multiple interactions between catechol groups/amino groups and tissues, the biocompatible hydrogels can strongly adhere on the surface of tissue. To further study, the bleeding rat-liver models are made to evaluate the hemostatic effects. After injecting the hydrogel precursor solution into the rat body, the hydrogels are not only formed in situ within 30 s but are also firmly adhered to the bleeding tissues which shows effective hemostasis. The injectability and tissue-adhesion improvement in this study gives a new insight into hemostatic agents, and the multifunctional hydrogels have a great potential in the biomedical application.

Biomimetic Bilayer Scaffold as an Incubator to Induce Sequential Chondrogenesis and Osteogenesis of Adipose Derived Stem Cells for Construction of Osteochondral Tissue.

Toward osteochondral tissue construction, the present study introduced a bilayer scaffold to induce sequential chondrogenesis and osteogenesis of stem cells in vitro. Two scaffolds that are both based on poly(l-glutamic acid) (PLGA) and chitosan (CS) were combined to form the bilayer scaffold. The cartilage region was the covalently cross-linked PLGA/CS hydrogel with a tubular pore structure, possessing a swollen network to prevent cellular adhesion, while inducing spontaneous cellular aggregate formation. The bone region was the electrostatically cross-linked PLGA-grafted nano hydroxyapatite (nHA-g-PLGA)/CS scaffold, which supported cellular adhesion and spreading. Human adipose derived stem cells (hASCs) were seeded into the cartilage region and observed to aggregate, formimg multicellular spheroids, which subsequently fused to rod-like aggregates with a larger size. At the same time, hASCs in aggregates crossed the interface and entered the bone region, presenting adhesion and spreading. With the induction of bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) during the first 14 days and BMP-2 alone during the last 14 days, hASCs aggregates in the cartilage region underwent chondrogenesis, expressing an abundant cartilage matrix including glycosaminoglycans (GAGs) and type II collagen (COL II) at 28 days. The chondrogenic induced hASCs migrated in the bone region turned to osteogenesis at 28 days, which was associated with their large spreading area and the switch of the induce factor. Thus, the present bilayer scaffold induced the different distribution of hASCs, resulting in subsequent chondrogenesis and osteogenesis, realizing osteochondral tissue construction in vitro.

Biodegradable High-Strength Hydrogels with Injectable Performance Based on Poly(l-Glutamic Acid) and Gellan Gum.

Currently, biodegradable hydrogels are one of the most promising materials in tissue engineering. From the perspective of clinical needs, hydrogels with high strength and minimally invasive implantation are preferred for the reconstruction of load-bearing tissues. In this work, a biodegradable, high-strength, and injectable hydrogel was developed by one-step photo-cross-linking of two biomacromolecules, polyethylene glycol acrylated poly(l-glutamic acid) (PLGA-APEG) and methacrylated gellan gum (GG-MA). The hydrogels, named as PLGA/GG hydrogels, exhibited high mechanical properties. The compression stress of the hydrogels was 0.53 MPa, and the fracture energy was 7.7 ± 0.2 kJ m-2. Meanwhile, the storage modulus could reach 44.0 ± 0.6 kPa. The hydrogel precursor solution loaded with adipose-derived stem cells (ASCs) was subcutaneously injected into C57BL/6 mice and then cross-linked via in situ transdermal irradiation, which demonstrated the excellent injectability and subcutaneous gelatinization of PLGA/GG hydrogels as cell carriers. Furthermore, three-dimensional encapsulation of ASCs in hydrogels after 7, 14, and 21 days showed outstanding cytocompatibility, and the viability of ASCs was up to 84.0 ± 1.7%. The PLGA/GG hydrogels exhibited ideal behaviors of degradation, with 60 ± 5% of hydrogels degraded in phosphate buffered solution (PBS) after 11 weeks. H&E staining demonstrated that the hydrogels degraded gradually after 6 weeks and supported tissue invasion without inflammatory reactions, which indicated the laudable biodegradability of hydrogels. Hence, the biodegradable and high-strength hydrogels with well-performed injectability and biocompatibility possessed high potential applications in tissue engineering, especially in load-bearing tissue regeneration.

Stack-Based Hydrogels with Mechanical Enhancement, High Stability, Self-Healing Property, and Thermoplasticity from Poly(l-glutamic acid) and Ureido-Pyrimidinone.

Supramolecular hydrogels formed by noncovalent bonds are attractive "smart" materials, which can rapidly respond to external stimuli. However, only a handful of supramolecular hydrogels is applicable in tissue engineering, due to the instability and poor mechanical strength of noncovalent cross-linking hydrogels. Thus, a rigid and stable supramolecular hydrogel has been developed based on poly(l-glutamic acid) and 2-ureido-4[1H]pyrimidinones (UPy), and the UPy stacks are noncovalent cross-linking interactions. The hydrogels show excellent mechanical strength and stability, in sharp contrast to noncovalent hydrogels cross-linked by UPy dimers and covalent hydrogels cross-linked by esterification. The hydrogels also exhibit remoldability, self-healing, and thermoplastic printing characteristics, which are caused by the reversible supramolecular property of UPy stacks. Also, the formation of hydrogels dependent on UPy stacks is further investigated by atomic force microscope, small-angle X-ray scattering, in situ X-ray diffraction, circular dichroism, and UV-vis spectroscopies. Finally, the hydrogels show commendable biocompatibility and degradability, which have high potential applications in regenerative medicine.

Nucleolar stress: is there a reverse version?

The nucleolus is a dynamic structure that has roles in various physiological and pathophysiological processes. Perturbations on many aspects of the nucleolar functions are thought to cause "nucleolar stress", which occurs in response to a variety of chemotherapeutic drugs. The main characteristic changes of nucleolar stress include: 1) reduction of the size and volume of the nucleolus; 2) inhibition of RNA Pol I-mediated rRNA synthesis; and 3) nucleoplasmic translocation of nucleolar stress-related proteins. In studying the apoptosis-inducing effect of the natural compound lovastatin (LV) on breast cancer stem cells, we unexpectedly uncovered a novel form of nucleolar stress, which we call "reverse nucleolar stress". In our system, the canonical nucleolus stress inducer doxorubicin caused nucleoplasmic translocation of the nucleolar protein NPM and complete abolishment of Nolc1, an NPM-interacting protein and an activator of rRNA transcription. In contrast, the reverse nucleolar stress induced by LV is manifested as a more localized perinucleolar distribution of NPM and an increase in the protein level of Nolc1. Furthermore, translocation of the ribosomal protein RPL3 from the cytoplasm to the nucleolus and increased AgNOR staining were observed. These changes characterize a novel pattern of nucleolar stress doubtlessly distinguishable from the canonical one. The functional consequences of reverse nucleolar stress are not clear at present but may presumably be related to cell death or even normalization of the stressed cell. The discovery of reverse nucleolar stress opens up a new area of research in molecular and cellular biology and might have important implications in cancer therapy.