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Osteoarthritis (OA) is the most prevalent cartilage degenerative and low-grade inflammatory disease of the whole joint. However, there are currently no FDA-approved drugs or global regulatory agency-approved treatments OA disease modification. Therefore, it's essential to explore novel effective therapeutic strategies for OA. In our study, we investigated the effects of AFK-PD, a novel pyridone agent, on the development of OA induced by destabilization of the medial meniscus (DMM) in vivo, and its impact on the function of chondrocytes treated with IL-1ß in vitro. Our results demonstrated AFK-PD alleviated OA progression through inhibiting cartilage degeneration, articular inflammation and osteophyte formation. Notably, AFK-PD inhibited chondrocyte inflammation and synovial macrophage M1 polarization, leading to the attenuation of articular inflammation. Additionally, AFK-PD promoted chondrocyte anabolism while mitigating catabolism and apoptosis, effectively inhibiting cartilage degeneration. Mechanistically, AFK-PD suppressed the expression of key signaling molecules involved in the MAPK pathway, such as p-ERK1/2 and p-JNK, as well as the NF-κB signaling molecule p-p65, in IL-1ß-induced chondrocytes. These findings suggest AFK-PD ameliorates the development of OA by protecting chondrocyte functions and inhibiting articular inflammation in chondrocytes and synovial macrophages. Overall, our study highlights AFK-PD as a promising therapeutic candidate for the treatment of OA.

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Diabetic bone defects, exacerbated by hyperglycemia-induced inflammation and oxidative stress, present significant therapeutic challenges. This study introduces a novel injectable scaffold, MgH2@PLGA/F-GM, consisting of foamed gelatin-methacryloyl (GelMA) and magnesium hydride (MgH2) microspheres encapsulated in poly(lactic-co-glycolic acid) (PLGA). This scaffold is uniquely suited for diabetic bone defects, conforming to complex shapes and fostering an environment conducive to tissue regeneration. As it degrades, Mg(OH)2 is released and dissolved by PLGA's acidic byproducts, releasing therapeutic Mg2+ ions. These ions are instrumental in macrophage phenotype modulation, inflammation reduction, and angiogenesis promotion, all vital for diabetic bone healing. Additionally, hydrogen (H2) released during degradation mitigates oxidative stress by diminishing reactive oxygen species (ROS). This multifaceted approach not only reduces ROS and inflammation but also enhances M2 macrophage polarization and cell migration, culminating in improved angiogenesis and bone repair. This scaffold presents an innovative strategy for addressing the complexities of diabetic bone defect treatment.

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Immunoregulatory and vascularized microenvironments play an important role in bone regeneration; however, the precise regulation for vascularization and inflammatory reactions remains elusive during bone repair. In this study, by means of subcutaneous preimplantation, we successfully constructed demineralized bone matrix (DBM) grafts with immunoregulatory and vascularized microenvironments. According to the current results, at the early time points (days 1 and 3), subcutaneously implanted DBM grafts recruited a large number of pro-inflammatory M1 macrophages with positive expression of CD68 and iNOS, while at the later time points (days 7 and 14), these inflammatory cells gradually subsided, accompanying increased presence of anti-inflammatory M2 macrophages with positive expression of CD206 and Arg-1, indicating a gradually enhanced anti-inflammatory microenvironment. At the same time, the gradually increased angiogenesis was observed in the DBM grafts with implantation time. In addition, the positive cells of CD105, CD73, and CD90 were observed in the inner region of the DBM grafts, implying the homing of mesenchymal stem cells. The repair results of cranial bone defects in a rat model further confirmed that the subcutaneous DBM xenografts at 7 days significantly improved bone regeneration. In summary, we developed a simple and novel strategy for bone regeneration mediated by anti-inflammatory microenvironment, prevascularization, and endogenous stem cell homing.

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Abdominal adhesions, a commonly observed complication of abdominal surgery, have a high incidence and adversely affect patients' physical and mental health. The primary causes of abdominal adhesions are intraoperative trauma, acute inflammatory response, bleeding, and foreign body infection. Because most current treatment approaches for abdominal adhesions are limited, improved and novel postoperative anti-adhesion regimens are urgently needed. In this study, we developed calcium polyphenol network (CaPN) microspheres based on the self-assembly of the natural triphenolic compound gallic acid and Ca2+ in solution. The physicochemical properties of CaPNs, including their hemostatic, antibacterial, antioxidant, and anti-inflammatory activities, were investigated in vitro. Bleeding and cecal-abdominal wall adhesion models were established to observe the hemostatic activity of CaPNs and their preventive effect on postoperative abdominal wall adhesion in vivo. The results showed that CaPNs significantly reduced inflammation, oxidative stress, fibrosis, and abdominal adhesion formation and had good hemostatic and antibacterial properties. Our findings suggest a novel strategy for the prevention of postoperative adhesions.

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Chinese hamster ovary cells are the main expression system for recombinant therapeutic proteins. During the production of these proteins, certain host cell proteins are secreted, broken down, and released by host cells in the culture along with the proteins of interest. These host cell proteins are often difficult to remove during the downstream purification process, and thus affect the quality, safety, and effectiveness of recombinant protein biopharmaceutical products and increase the production cost of recombinant therapeutic proteins. Therefore, host cell protein production must be reduced as much as possible during the production process and eliminated during purification. This article reviews the harm caused by host cell proteins in the production of recombinant protein drugs using Chinese hamster ovary cell, factors affecting host cell proteins, the monitoring and identification of these proteins, and methods to reduce their type and quantity in the final product.

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Human decalcified bone matrix (HDBM) is a framework with a porous structure and good biocompatibility. Nevertheless, its oversized pores lead to massive cell loss when seeding chondrocytes directly over it. Gelatin (GT) is a type of protein obtained by partial hydrolysis of collagen. The GT scaffold can be prepared from the GT solution through freeze-drying. More importantly, the pore size of the GT scaffold can be controlled by optimizing the concentration of the GT solution. Similarly, when different concentrations of gelatin are combined with HDBM and then freeze-dried, the pore size of the HDBM can be modified to different degrees. In this study, the HDBM framework was modified with 0.3, 0.6, and 0.9%GT, resulting in an improved pore size and adhesion rate. Results showed that the HDBM framework with 0.6%GT (HDBM-0.6%GT) had an average pore size of 200 µm, which was more suitable for chondrocyte seeding. Additionally, our study validated that porcine decalcified bone matrix (PDBM) had a proper pore structure. Chondrocytes were in vitro seeded on the three frameworks for 4 weeks and then implanted in nude mice and autologous goats, respectively. The in vivo cartilage regeneration results showed that HDBM-0.6%GT and PDBM frameworks compensated for the oversized pores of the HDBM framework. Moreover, they showed successfully regenerated more mature cartilage tissue with a certain shape in animals.

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Three-dimensional (3D) bioprinted cartilage-mimicking substitutes for full-thickness articular cartilage defect repair have emerged as alternatives to in situ defect repair models. However, there has been very limited breakthrough in cartilage regeneration based on 3D bioprinting owing to the lack of ideal bioinks with printability, biocompatibility, bioactivity, and suitable physicochemical properties. In contrast to animal-derived natural polymers or acellular matrices, human-derived Wharton's jelly is biocompatible and hypoimmunogenic with an abundant source. Although acellular Wharton's jelly can mimic the chondrogenic microenvironment, it remains challenging to prepare both printable and biologically active bioinks from this material. Here, we firstly prepared methacryloyl-modified acellular Wharton's jelly (AWJMA) using a previously established photo-crosslinking strategy. Subsequently, we combined methacryloyl-modified gelatin with AWJMA to obtain a hybrid hydrogel that exhibited both physicochemical properties and biological activities that were suitable for 3D bioprinting. Moreover, bone marrow mesenchymal stem cell-loaded 3D-bioprinted cartilage-mimicking substitutes had superior advantages for the survival, proliferation, spreading, and chondrogenic differentiation of bone marrow mesenchymal stem cells, which enabled satisfactory repair of a model of full-thickness articular cartilage defect in the rabbit knee joint. The current study provides a novel strategy based on 3D bioprinting of cartilage-mimicking substitutes for full-thickness articular cartilage defect repair.

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Articular cartilage (AC), a bone-to-bone protective device made of up to 80% water and populated by only one cell type (i.e. chondrocyte), has limited capacity for regeneration and self-repair after being damaged because of its low cell density, alymphatic and avascular nature. Resulting repair of cartilage defects, such as osteoarthritis (OA), is highly challenging in clinical treatment. Fortunately, the development of tissue engineering provides a promising method for growing cells in cartilage regeneration and repair by using hydrogels or the porous scaffolds. In this paper, we review the therapeutic strategies for AC defects, including current treatment methods, engineering/regenerative strategies, recent advances in biomaterials, and present emphasize on the perspectives of gene regulation and therapy of noncoding RNAs (ncRNAs), such as circular RNA (circRNA) and microRNA (miRNA).

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Osteoarthritis (OA) is a chronic osteoarthropathy characterized by the progressive degeneration of articular cartilage and synovial inflammation. Early OA clinical treatments involve intra-articular injection of glucocorticoids, oral acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs), which are used for anti-inflammation and pain relief. However, long-term use of these agents will lead to inevitable side effects, even aggravate cartilage loss. At present, there are no disease-modifying OA drugs (DMOADs) yet approved by regulatory agencies. Polarization regulation of synovial macrophages is a new target for OA treatment. Inhibiting M1 polarization and promoting M2 polarization of synovial macrophages can alleviate synovial inflammation, relieve joint pain and inhibit articular cartilage degradation, which is a promising strategy for OA treatment. In this study, we describe the molecular mechanisms of macrophage polarization and its key role in the development of OA. Subsequently, we summarize the latest progress of strategies for OA treatment through macrophage reprogramming, including small molecule compounds (conventional western medicine and synthetic compounds, monomer compounds of traditional Chinese medicine), biomacromolecules, metal/metal oxides, cells, and cell derivatives, and interprets the molecular mechanisms, hoping to provide some information for DMOADs development.

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Recombinant therapeutic proteins (RTPs) are important parts of biopharmaceuticals. Chinese hamster ovary cells (CHO) have become the main cell hosts for the production of most RTPs approved for marketing because of their high-density suspension growth characteristics, and similar human post-translational modification patterns et al. In recent years, many studies have been performed on CHO cell expression systems, and the yields and quality of recombinant protein expression have been greatly improved. However, the expression levels of some proteins are still low or even difficult-to express in CHO cells. It is urgent further to increase the yields and to express successfully the "difficult-to express" protein in CHO cells. The process of recombinant protein expression of is a complex, involving multiple steps such as transcription, translation, folding processing and secretion. In addition, the inherent characteristics of molecular will also affect the production of protein. Here, we reviewed the factors affecting the expression of recombinant protein and improvement strategies in CHO cells.

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Plasma glutathione peroxidase (GPx3) belongs to the GPx superfamily, and it is the only known secreted selenocysteine (Sec)-containing GPx in humans. It exists as a glycosylated homotetramer and catalyzes the reduction of hydrogen peroxide and lipid peroxides, depending on the Sec in its active center. In this study, a previously reported chimeric tRNAUTuT6 was used for the incorporation of Sec at the UAG amber codon, and the mature form of human GPx3 (hGPx3) without the signal peptide was expressed in amber-less E. coli C321.ΔA.exp. Reactive Sec-hGPx3, able to reduce H2O2 and tert-butyl hydroperoxide (t-BuOOH), was produced with high purity and yield. Study of the quaternary structure suggested that the recombinant Sec-hGPx3 contained an intra-molecular disulfide bridge but failed to form tetramer. Mutational and structural analysis of the mutants with three Cys residues, individually or jointly replaced with Ser, indicated that the formation of intra-molecular disulfide bridges involved structure conformational changes. The secondary structure containing Cys77 and Cys132 was flexible and could form a disulfide bond, or form a sulfhydryl-selenyl bond with Sec49 in relative mutants. Mutation of Cys8 and Cys132 to Sec8 and Sec132 could fix the oligomerization loop through the formation of diselenide bond, which, in turn, facilitated tetramer formation and noticeably improved the GPx activity. This research provides an important foundation for the further catalysis and functional study of hGPx3.

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Chinese hamster ovary (CHO) cells are currently the most widely used host cells for production of recombinant therapeutic proteins (RTPs). Small-molecule additives related to cell cycle apoptosis and autophagy regulation have been used to promote RTP production. By combining two small-molecule additives, positive synergistic effects on transgene expression were observed in CHO cells. In the present study, six small-molecule additives were used, including hydrocinnamic acid (HCA), sodium butyrate (NaB), lithium acetate (LiAc), sodium succinate dibasic hexahydrate (SDH), decitabine (DAC), and sodium propionate (SP). Experiments to test the effects of their pairwise combinations on two different recombinant CHO cell lines (rCHO) were designed using Design-Expert 12.0. Different effects of various pairs of small molecules on apoptosis- and autophagy-related protein expression were observed in the rCHOs. The results showed that compared to the control culture, NaB alone increased the volumetric yield and specific productivity (Qp) by 166% and 143%, respectively. The volumetric yield and Qp of NaB combined with DAC (Cg1)-treated cells increased by 178% and 212%, respectively. Cg1 selectively blocked the cells in the G0/G1 cell cycle stage. The relative expression levels of B-cell lymphoma 2 (Bcl-2), Beclin 1, and microtubule-associated protein light chain 3 (LC3B) in Cg1-treated CHO cells were significantly increased, while relative levels of cleaved caspase-3 expression were significantly decreased. In conclusion, Cg1 had the most obvious effect on RTP production and Qp in CHO cells, suggesting the Cg1 combination of small molecules may be used to improve the expression of recombinant protein in CHO cells.

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As the most widely used mammalian cell line, Chinese hamster ovary (CHO) cells can express various recombinant proteins with a post translational modification pattern similar to that of the proteins from human cells. During industrial production, cells need large amounts of ATP to support growth and protein expression, and since glycometabolism is the main source of ATP for cells, protein production partly depends on the efficiency of glycometabolism. And efficient glycometabolism allows less glucose uptake by cells, reducing production costs, and providing a better mammalian production platform for recombinant protein expression. In the present study, a series of progresses on the comprehensive optimization in CHO cells by glycometabolism strategy were reviewed, including carbohydrate intake, pyruvate metabolism and mitochondrial metabolism. We analyzed the effects of gene regulation in the upstream and downstream of the glucose metabolism pathway on cell's growth and protein expression. And we also pointed out the latest metabolic studies that are potentially applicable on CHO cells. In the end, we elaborated the application of metabolic models in the study of CHO cell metabolism.

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At present, nearly 70% of recombinant therapeutic proteins (RTPs) are produced by Chinese hamster ovary (CHO) cells, and serum-free medium (SFM) is necessary for their culture to produce RTPs. In this review, the history and key components of SFM are first summarized, and its preparation and experimental design are described. Some small molecule compound additives can improve the yield and quality of RTP. The function and possible mechanisms of these additives are also reviewed here. Finally, the future perspectives of SFM use with CHO cells for RTP production are discussed.

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The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNAUTu) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321.ΔA.exp using tRNAUTu and seven chimeric tRNAs that were constructed on the basis of tRNAUTu. We found that chimeric tRNAUTu2, which substitutes the acceptor stem and T-stem of tRNAUTu with those from tRNASec, enabled the expression of reactive hGPx with high yields. We also found that chimeric tRNAUTuT6, which has a single base change (A59C) compared to tRNAUTu, mediated the highest reactive expression of hGPx1. The hGPx1 expressed exists as a tetramer and reacts with positive cooperativity. The SDS-PAGE analysis of hGPx2 produced by tRNAUTuT6 with or without sodium selenite supplementation showed that the incorporation of Sec is nearly 90%. Our approach enables efficient selenoprotein expression in amber-less Escherichia coli and should enable further characterization of selenoproteins in vitro.

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BACKGROUND: Medical disputes have become a serious issue in China. A crisis cannot usually be predicted and managed through a cost-benefit strategy; therefore, researchers believe that prevention is better than containment and post-crisis resolution. This study aimed to identify solutions to prevent medical disputes in surgical cases through early warning and intervention of potential cases. METHODS: A case-control study was conducted to identify early detection indicators of medical disputes in the surgical treatment of liver cancer through Delphi consultation and logistic regression on the basis of which interventions were undertaken to prevent potential cases. RESULTS: The dispute detection model was composed of patient age (P = 0.08), frequency of hospitalization (P = 0.003), length of hospital stay (P < 0.001), terminal condition (P = 0.004), unplanned reoperation (P = 0.048), blood transfusion volume (P = 0.006), and arrearage (P < 0.001). Risk management interventions through quality improvement and enhanced communication in cases with an abnormal performance indicator proved effective in practice. CONCLUSIONS: This study explored the use of an evidence-based medical risk management strategy for medical disputes that involved early detection and intervention and could potentially be adopted by hospitals to prevent medical disputes.

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Gold nanoparticles (GNPs) have considerable applications in biomedicine, such as in bio-sensing, bio-imaging, drug delivery and photothermal therapeutics. However, currently there are limited information regarding the impact of pregnancy on their biodistribution, elimination and toxicity. In this study, we investigated the biodistribution and potential toxic effects of different-sized GNPs (1.5, 4.5, 13, 30 and 70 nm in diameter) in non-pregnant and pregnant mice at different gestational ages (E5.5, 7.5, 9.5, 11.5 and 13.5). 5h after intravenous injection, GNPs exhibited size-dependent biodistribution profiles; however, regardless of size, no significant biodistribution changes were observed between non-pregnant and pregnant mice. Kinetic studies showed that 4.5 nm GNPs were primarily excreted through urine within 5h, whereas 30 nm GNPs had a more prolonged blood circulation time. No apparent toxic effects (e.g., increased mortality, altered behavior, reduced animal weight, abnormal organ morphology or reduced pregnancy duration) were observed with different-sized GNPs in pregnant mice. However, treatment with 30 nm GNPs induced mild emphysema-like changes in lungs of pregnant mice. These results indicated that the maternal biodistribution patterns of GNPs in pregnant mice depended on particle size, but not gestational age; organ-specific adverse effects may arise with treatment with some GNPs according to their size.

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Phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC1.11.1.12) is a selenoperoxidase that can directly reduce phospholipid and cholesterol hydroperoxides. The mature cytoplasmic GPx4 is a monomeric protein with molecular weight of 19.5 kDa. In this study, human GPx4 (hGPx4) gene was amplified from the complementary DNA library of human hepatoma cell line. Eukaryotic expression plasmid pSelExpress1-leader-GPx4 was constructed and transfected into the eukaryotic cells HEK293T. Expression of hGPx4 was detected by Western blotting, and the target protein was purified by immobilized metal affinity chromatography. The results of the activity and kinetics of the purified protein show that the obtained protein follows a "ping-pong" mechanism, which is similar to that of native cytosolic glutathione peroxidase (GPx1; EC1.11.1.9). This is the first time that hGPx4 could be expressed and purified from HEK293T cells, and this work will provide an important resource of hGPx4 for its functional study in vitro and in vivo.

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The materno-fetal transfer of nanoparticles is a critical issue in designing theranoustic nanoparticles for in vivo applications during pregnancy. Recent studies have reported that certain nanoparticles can cross the placental barrier in healthy pregnant animals depending on the size and surface modification of the nanoparticles and the developmental stages of the fetuses. However, materno-fetal transfer under pathological pregnant conditions has not been examined so far. Here, it is shown that intrauterine inflammation can enhance the materno-fetal transfer of nanoparticles in the late gestation stage of murine pregnancy in a size-dependent manner. Three different-sized gold nanoparticles (Au NPs) with diameters of 3 (Au3), 13 (Au13) and 32 (Au32) nm are applied. The accumulation of Au3 and Au13 nanoparticles in the fetuses is significantly increased in intrauterine inflammatory mice compared with healthy control mice: the concentration of Au3 is much higher than Au13 in fetal tissues of intrauterine inflammatory mice. In contrast, Au32 nanoparticles cannot cross the placental barrier either in healthy or in intrauterine inflammatory mice. The possible underlying mechanism of the increased materno-fetal transfer of small-sized nanoparticles on pathological conditions is inferred to be the structural and functional abnormalities of the placenta under intrauterine inflammation. The size of the nanoparticles is one of the critical factors which determines the extent of fetal exposure to nanoparticles in murine pregnancy under inflammatory conditions.

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Nanoparticle exposure in pregnancy may result in placental damage and fetotoxicity; however, the factors that determine fetal nanoparticle exposure are unclear. Here we have assessed the effect of gestational age and nanoparticle composition on fetal accumulation of maternally-administered nanomaterials in mice. We determined the placental and fetal uptake of 13 nm gold nanoparticles with different surface modifications (ferritin, PEG and citrate) following intravenous administration at E5.5-15.5. We showed that prior to E11.5, all tested nanoparticles could be visualized and detected in fetal tissues in significant amounts; however, fetal gold levels declined dramatically post-E11.5. In contrast, Au-nanoparticle accumulation in the extraembryonic tissues (EET) increased 6-15 fold with gestational age. Fetal and EET accumulation of ferritin- and PEG-modified nanoparticles was considerably greater than citrate-capped nanoparticles. No signs of toxicity were observed. Fetal exposure to nanoparticles in murine pregnancy is, therefore, influenced by both stage of embryonic/placental maturation and nanoparticle surface composition.

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