Application of Bioactive Materials for Osteogenic Function in Bone Tissue Engineering.

Bone tissue defects present a major challenge in orthopedic surgery. Bone tissue engineering using multiple versatile bioactive materials is a potential strategy for bone-defect repair and regeneration. Due to their unique physicochemical and mechanical properties, biofunctional materials can enhance cellular adhesion, proliferation, and osteogenic differentiation, thereby supporting and stimulating the formation of new bone tissue. 3D bioprinting and physical stimuli-responsive strategies have been employed in various studies on bone regeneration for the fabrication of desired multifunctional biomaterials with integrated bone tissue repair and regeneration properties. In this review, biomaterials applied to bone tissue engineering, emerging 3D bioprinting techniques, and physical stimuli-responsive strategies for the rational manufacturing of novel biomaterials with bone therapeutic and regenerative functions are summarized. Furthermore, the impact of biomaterials on the osteogenic differentiation of stem cells and the potential pathways associated with biomaterial-induced osteogenesis are discussed.

The Generation of a Nanobody-Based ELISA for Human Microsomal Epoxide Hydrolase.

A microsomal epoxide hydrolase (mEH) metabolizes in vivo in both xenobiotic and endogenous epoxides associated with signaling function. Findings in patients suggest that mEH might be a biomarker for several diseases, including metastatic cancer and viral hepatitis. To easily quantify mEH, nanobodies specific to the human mEH were isolated from a phage library of llama VHHs. Four unique clones were obtained and used for developing ELISAs. Three formats of double antibody sandwich assays were investigated using different detection strategies. Using PolyHRP, the signal was strongly amplified, yielding a 22-fold lower LOD (12 pg mL-1) than the 'conventional'. To further validate the performance of the immunoassays, human tissue samples were analyzed by nanobody-based ELISAs and compared to the enzyme activities (R2 > 0.95). The results demonstrate that these nanobodies are powerful tools for the quantification of human mEH and could eventually result in a bedside assay.

Design, Synthesis, and Evaluation of Glucose Transporter Inhibitor-SN38 Conjugates for Targeting Colorectal Cancer.

Irinotecan (1), a prodrug of SN38 (2) approved by the US Food and Drug Administration for treating colorectal cancer, lacks specificity and causes many side effects. To increase the selectivity and therapeutic efficacy of this drug, we designed and synthesized conjugates of SN38 and glucose transporter inhibitors (phlorizin (5) or phloretin (6)), which could be hydrolyzed by glutathione or cathepsin to release SN38 in the tumor microenvironment, as a proof of concept. These conjugates (8, 9, and 10) displayed better antitumor efficacy with lower systemic exposure to SN38 in an orthotopic colorectal cancer mouse model compared with irinotecan at the same dosage. Further, no major adverse effects of the conjugates were observed during treatment. Biodistribution studies showed that conjugate 10 could induce higher concentrations of free SN38 in tumor tissues than irinotecan at the same dosage. Thus, the developed conjugates exhibit potential for treating colorectal cancer.

Low-intensity pulsed ultrasound promotes mesenchymal stem cell transplantation-based articular cartilage regeneration via inhibiting the TNF signaling pathway.

BACKGROUND: Mesenchymal stem cell (MSC) transplantation therapy is highly investigated for the regenerative repair of cartilage defects. Low-intensity pulsed ultrasound (LIPUS) has the potential to promote chondrogenic differentiation of MSCs. However, its underlying mechanism remains unclear. Here, we investigated the promoting effects and mechanisms underlying LIPUS stimulation on the chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) and further evaluated its regenerative application value in articular cartilage defects in rats. METHODS: LIPUS was applied to stimulate cultured hUC-MSCs and C28/I2 cells in vitro. Immunofluorescence staining, qPCR analysis, and transcriptome sequencing were used to detect mature cartilage-related markers of gene and protein expression for a comprehensive evaluation of differentiation. Injured articular cartilage rat models were established for further hUC-MSC transplantation and LIPUS stimulation in vivo. Histopathology and H&E staining were used to evaluate the repair effects of the injured articular cartilage with LIPUS stimulation. RESULTS: The results showed that LIPUS stimulation with specific parameters effectively promoted the expression of mature cartilage-related genes and proteins, inhibited TNF-α gene expression in hUC-MSCs, and exhibited anti-inflammation in C28/I2 cells. In addition, the articular cartilage defects of rats were significantly repaired after hUC-MSC transplantation and LIPUS stimulation. CONCLUSIONS: Taken together, LIPUS stimulation could realize articular cartilage regeneration based on hUC-MSC transplantation due to the inhibition of the TNF signaling pathway, which is of clinical value for the relief of osteoarthritis.

Injectable self-assembled dual-crosslinked alginate/recombinant collagen-based hydrogel for endometrium regeneration.

The disadvantages of mainstream therapies for endometrial injury are difficult to resolve, herein, we suggest an omnibearing improvement strategy by introducing an injectable multifunctional self-assembled dual-crosslinked sodium alginate/recombinant collagen hydrogel. The hydrogel possessed a reversible and dynamic double network based on dynamic covalent bonds and ionic interactions, which also contributed to excellent capability in viscosity and injectability. Moreover, it was also biodegradable with a suitable speed, giving off active ingredients during the degradation process and eventually disappearing completely. In vitro tests exhibited that the hydrogel was biocompatible and able to enhance endometrial stromal cells viability. These features synergistically promoted cell multiplication and maintenance of endometrial hormone homeostasis, which accelerated endometrial matrix regeneration and structural reconstruction after severe injury in vivo. Furthermore, we explored the interrelation between the hydrogel characteristics, endometrial structure, and postoperative uterine recovery, which would benefit deep research on regulation of uterine repair mechanism and optimization of hydrogel materials. The injectable hydrogel could achieve favourable therapeutic efficacy without the need of exogenous hormones or cells, which would be of clinical value in endometrium regeneration.

Generation of bioluminescent enzyme immunoassay for ferritin by single-chain variable fragment and its NanoLuc luciferase fusion.

Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

rBMSC osteogenic differentiation enhanced by graphene quantum dots loaded with immunomodulatory layered double hydroxide nanoparticles.

Bone tissue defects caused by disease, trauma, aging or genetic factors emerged as one of the main factors that endanger human health. At present, advanced development of bone tissue engineering and regenerative medicine focused on the biomaterials regulated stem cell for responsive differentiation.In vivotransplantation of allogeneic bone materials has the needs of both osteogenic and immune regulation function. In this study, we utilized the extensively proved biocompatible layered double hydroxide (LDH) nanoparticles as the nanocarrier of graphene quantum dots (GQD), the functional loading was validated by characteristics analysis of scanning electron microscopy, surface zeta potential, X-ray diffraction and fourier transform infrared spectroscopy. Further, we investigated the cellular uptake of nanoparticles in rat bone marrow derived mesenchymal stem cells, the significant enhanced endocytosis was occurred in LDH-GQD treated groups. The enhanced osteogenic differentiation abilities of LDH-GQD were systematically investigated through alkaline phosphatase staining, alizarin red staining and qPCR analysis. In addition, the anti-inflammatory regulation of LDH facilitated the phenotypic transition of macrophage in LDH-GQD nanocomposites. Overall, the successful construction and functional validation of nanomaterials in this study will provide clinical therapeutic potential in bone defects regeneration.

A highly sensitive electrochemical biosensor for microRNA122 detection based on a target-induced DNA nanostructure.

Specific and sensitive biomarker detection is significant for the early diagnosis of cancers. Herein, a highly sensitive electrochemical biosensor employing a tetrahedral DNA nanostructure (TDN) probe and multiple signal amplification strategies has been constructed, and successfully applied to microRNA-122 (miR-122) detection. The platform consisted of a TDN probe anchoring on a gold nanoparticle-coated gold electrode and multiple signal amplification procedures combining the electrodeposition of gold nanoparticles, hybridization chain reaction (HCR), and horseradish peroxidase enzymatic catalysis (HPEC). In the presence of the target, the hairpin structure of the helper probe could be opened and trigger the HCR through the hybridization of H1 and H2 probes, and then avidin-HRP was attached on the surface of the gold electrode that can produce an electro-catalytic signal. We used TDN probe as the scaffold to increase the reactivity and multiple signal amplification greatly improve the sensitivity of this biosensor. This biosensor offers an excellent sensitivity (a limit of detection of 0.74 aM) and differentiation ability for single and multiple mismatches. This multiplexing biosensor for trace microRNA detection shows promising applications in the early diagnosis of cancer.

Enhanced performance of a surface plasmon resonance-based immunosensor for the detection of glycocholic acid.

The concentration of glycocholic acid (GCA) in urine and blood is an important biomarker for liver cancer. Monitoring of GCA depends to a large extent on the availability of appropriate analytical techniques. In this work, based on the immobilization of GCA-OVA onto the sensor chip surface, a label-free competitive inhibition immunoassay for the determination of GCA with the surface plasmon resonance (SPR) technique was developed. The proposed SPR immunosensor is simple to prepare, recyclable and exhibits excellent sensitivity to GCA (a linear range of 13.3-119.4 ng mL-1 and a limit of detection (LOD) of 2.5 ng mL-1), which was 14 times lower than that of the traditional immunoassay. Excellent recoveries and correlation between these two methods were observed (R2 = 0.995). Hence, it can be proved that the SPR immunosensor could be used to achieve rapid and sensitive quantitative detection of GCA in real urine samples and meet clinical needs.

Targeting Colorectal Cancer with Conjugates of a Glucose Transporter Inhibitor and 5-Fluorouracil.

Overexpression of glucose transporters (GLUTs) in colorectal cancer cells is associated with 5-fluorouracil (1, 5-FU) resistance and poor clinical outcomes. We designed and synthesized a novel GLUT-targeting drug conjugate, triggered by glutathione in the tumor microenvironment, that releases 5-FU and GLUTs inhibitor (phlorizin (2) and phloretin (3)). Using an orthotopic colorectal cancer mice model, we showed that the conjugate exhibited better antitumor efficacy than 5-FU, with much lower exposure of 5-FU during treatment and without significant side effects. Our study establishes a GLUT-targeting theranostic incorporating a disulfide linker between the targeting module and cytotoxic payload as a potential antitumor therapy.

A Type IIb, but Not Type IIa, GnRH Receptor Mediates GnRH-Induced Release of Growth Hormone in the Ricefield Eel.

Multiple gonadotropin-releasing hormone receptors (GnRHRs) are present in vertebrates, but their differential physiological relevances remain to be clarified. In the present study, we identified three GnRH ligands GnRH1 (pjGnRH), GnRH2 (cGnRH-II), and GnRH3 (sGnRH) from the brain, and two GnRH receptors GnRHR1 (GnRHR IIa) and GnRHR2 (GnRHR IIb) from the pituitary of the ricefield eel Monopterus albus. GnRH1 and GnRH3 but not GnRH2 immunoreactive neurons were detected in the pre-optic area, hypothalamus, and pituitary, suggesting that GnRH1 and GnRH3 may exert hypophysiotropic roles in ricefield eels. gnrhr1 mRNA was mainly detected in the pituitary, whereas gnrhr2 mRNA broadly in tissues of both females and males. In the pituitary, GnRHR1 and GnRHR2 immunoreactive cells were differentially distributed, with GnRHR1 immunoreactive cells mainly in peripheral areas of the adenohypophysis whereas GnRHR2 immunoreactive cells in the multicellular layers of adenohypophysis adjacent to the neurohypophysis. Dual-label fluorescent immunostaining showed that GnRHR2 but not GnRHR1 was localized to somatotropes, and all somatotropes are GnRHR2-positive cells and vice versa at all stages examined. GnRH1 and GnRH3 were shown to stimulate growth hormone (Gh) release from primary culture of pituitary cells, and to decrease Gh contents in the pituitary of ricefield eels 12 h post injection. GnRH1 and GnRH3 stimulated Gh release probably via PLC/IP3/PKC and Ca2+ pathways. These results, as a whole, suggested that GnRHs may bind to GnRHR2 but not GnRHR1 to trigger Gh release in ricefield eels, and provided novel information on differential roles of multiple GnRH receptors in vertebrates.

Analysis of the hormone receptor status of circulating tumor cell subpopulations based on epithelial-mesenchymal transition: a proof-of-principle study on the heterogeneity of circulating tumor cells.

Although the enumeration of circulating tumor cells (CTCs) has been demonstrated to be a prognostic indicator in metastatic breast cancer, the heterogeneous characteristics of CTCs, such as variations in the epithelial-mesenchymal transition (EMT), may limit its broad clinical application. To investigate an uncomplicated and practicable detection approach based on the potential utility of the heterogeneity of CTCs from the standpoint of the EMT phenotype and ER/PR status of CTCs, an analysis was conducted using peripheral blood samples obtained from 28 metastatic breast cancer patients. The CanPatrol CTC enrichment technique was used to identify different CTC subpopulations, including epithelial-dominated CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal-dominated CTCs, according to epithelial and mesenchymal markers. Furthermore, the hormone receptor (HR) status of each CTC was determined based on the expression levels of three reference genes and was characterized by four levels, which ranged from high-level expression to non-expression. We subsequently concluded that based on EMT phenotypes, the order of different CTC subgroups differed according to the HR expression status of the primary tumor. With respect to the HR status between tissues and CTCs, the variation tendency from high-level expression to non-expression of HR in CTCs was significantly correlated with the HR status of the primary tumor. The findings could provide evidence for the potential application of this uncomplicated and practicable detection approach for prognostic analysis and individualized endocrine therapeutic direction in a real-time manner via confirmation in further large-scale trials.

[Detection and Analysis of EGFR and KRAS Mutation with Lung Adenocarcinoma].

BACKGROUND AND OBJECTIVE: Mutations in epidermal growth factor receptor (EGFR) and KRAS are important markers in non-small cell lung cancer, which are closely related to the clinical therapeutic effect. To analysis the EGFR and KRAS gene mutation rate and its relationship with clinical features in patients with lung adenocarcinoma. METHODS: 395 patients with treatment naïve lung adenocarcinoma, tumor tissue samples were available for testing. Tumor sample EGFR and KRAS mutation status were detected using mutant enriched liquidchip. RESULTS: 395 cases of lung adenocarcinoma, EGFR mutations were detected in 192 cases (48.9%), KRAS mutations were detected in 29 cases (7.8%), and the presence of EGFR and KRAS mutation were detected in 1 case (0.3%). EGFR mutations were found to occur significantly more often in female than in male patients (62.0% vs 37.1%, P<0.001) and in never smokers than in smokers (61.9% vs 30.3%, P<0.001), no significant differences were observed in age, stage and different biopsy type. KRAS mutations were not found to have statistical significance (P>0.05) in each clinical factors, only occurred in the wild type EGFR gene in patients (13.5%, 27/200) was significantly higher than that of patients with EGFR mutation (1.0%, 2/192), the difference was statistically significant (P<0.001). CONCLUSIONS: In lung adenocarcinomas, EGFR mutation was higher in female and non-smoking patients, KRAS mutation only in patients with wild-type EGFR gene was higher. Before using TKI targeted therapy, EGFR and KRAS mutations should be detected.


[Detection and Analysis of EGFR and KRAS Mutations 
in the Patients with Lung Squamous Cell Carcinomas].

BACKGROUND: Activating mutations in epidermal growth factor receptor (EGFR) and KRAS are important markers in non-small cell lung cancer. However, EGFR and KRAS gene mutations in lung squamous cell carcinoma are rarely reported. The aim of this study was to analyze EGFR and KRAS gene mutation rate and their relationship with clinical features in patients with lung squamous cell carcinomas. METHODS: A total of 139 patients undergoing treatment for naïve lung squamous cell carcinomas with tumor tissue samples available for testing were recruited. EGFR and KRAS mutation statuses of the tumor samples were detected using a mutant enriched liquid chip. RESULTS: Of the 139 cases of lung squamous cell carcinoma, EGFR mutations were detected in 25 cases (18%), KRAS mutations were detected in 7 cases (5%), and the presence of both EGFR and KRAS mutations was detected in 1 case (0.7%). EGFR mutations occurred more often in females than in males (33.3% vs 16.5%) and in patients that never smoked than in those who smoke (29.6% vs 16.1%). However, the difference did not reach statistical significance (P>0.05). No significant differences were observed in age, stage, and different biopsy type. KRAS mutations occurred more often in males than in females (5.5% vs 0%), but the difference did not reach statistical significance (P>0.05). No significant differences were observed in age, stage, different biopsy type, and smoking status (P>0.05). CONCLUSIONS: EGFR and KRAS mutations were low in lung squamous cell carcinomas, and had no significant correlation with clinical features. Before using tyrosine kinase inhibitor targeted therapy, EGFR and KRAS mutations should be detected in patients with lung squamous cell carcinomas.

Identifying EGFR mutations from SCLC patient plasma by mutant-enriched liquidchip technology.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as erlotinib and gefitinib are targeted drugs for the kinase domain of EGFR. They are widely used for the treatment of non-small cell lung cancer (NSCLC). The EGFR exon 19 deletion mutation and the L858R mutation in exon 21 comprise approximately 90% of the somatic mutations in NSCLC patients that respond to EGFR TKI. Several recent studies have also reported that small cell lung cancer (SCLC) patients with EGFR mutations responded to gefitinib. Further study, however, has been limited due to the difficulty obtaining tumor specimens from SCLC patients. OBJECTIVES: The aim of this study was to explore the EGFR mutation status in SCLC patients by plasma analysis. MATERIAL AND METHODS: Plasma samples from SCLC patients were collected for mutant-enriched liquidchip (MEL) analysis to identify the EGFR mutations in exon 19 and 21. RESULTS: The exon 19 deletion mutation was detected in one out of 35 patients (a female non-smoker). No exon 21 mutations were found. CONCLUSIONS: A prevalence of EGFR mutations in SCLC is rare, and occurs most frequently in females and nonsmokers.

Comparison of EGFR signaling pathway somatic DNA mutations derived from peripheral blood and corresponding tumor tissue of patients with advanced non-small-cell lung cancer using liquidchip technology.

Somatic DNA mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway are known to predict responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers. We evaluated a sensitive liquidchip platform for detecting EGFR, KRAS (alias Ki-ras), proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations in plasma samples, which were highly correlated with matched tumor tissues from 86 patients with advanced non-small-cell lung cancers. Either EGFR exon 19 or 21 mutations were detected in 36 patients: 23 of whom had identical mutations in both their blood and tissue samples; whereas mutations in the remaining 13 were found only in their tumor samples. These EGFR mutations occurred at a significantly higher frequency in females, never-smokers, and in patients with adenocarcinomas (P ≤ 0.001). The EGFR exon 20 T790M mutation was detected in only one of the paired samples [100% (95% CI, 96% to 100%) agreement]. For KRAS, proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations, the overall agreements were 97% (95% CI, 90% to 99%), 98% (95% CI, 92% to 99%), and 97% (95% CI, 90% to 99%), respectively, and these were not associated with age, sex, smoking history, or histopathologic type. In conclusion, mutations detected in plasma correlated strongly with mutation profiles in each respective tumor sample, suggesting that this liquidchip platform may offer a rapid and noninvasive method for predicting tumor responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers.

Exploratory study on the correlation between 14 lung cancer-related gene expression and specific clinical characteristics of NSCLC patients.

Personalized medicine has become essential in the treatment of lung cancer. However, the lung cancer-related gene expression profiles in non-small cell lung cancer (NSCLC) patients have not been elucidated. In this study, the correlation between gene expression profiles and clinicopathological characteristics was investigated in NSCLC patients. A total of 95 patients were enrolled in this study. The mRNA expression levels of 14 genes were assessed by multiplex branched DNA liquidchip (MBL) technology and data on 9 clinicopathological characteristics of patients were collected simultaneously. The correlation between gene expression and clinicopathological characteristics was investigated. Out of the 9 clinicopathological parameters, 6 were associated with several of the 14 genes analyzed. Patient gender was associated with TYMS and TOP2A. Clinical stage was associated with VEGFR2, KIT and HER2. There was weak correlation between primary tumor size of ≤3 cm and the expression level of KIT. The mRNA expression levels of VEGFR2 and HER2 correlated with distant metastasis. BRCA1, TYMS, TOP2A and HER2 were associated with histological type. Smoking correlated with higher expression levels of BRCA1, TYMS and TOP2A and lower expression levels of PDGFRß. The results were suggestive of correlation between the clinicopathological parameters of the NSCLC patients and the mRNA expression levels of certain lung cancer-related genes, including BRCA1, TYMS, TOP2A, PDGFRß, VEGFR2, KIT and HER2.

Personalized medicine of esophageal cancer.

The fatality rate of esophageal carcinomas is high in developing countries, making effective treatment desirable. Traditional treatment has now entered into the platform, and treatments based on the detection of biomarkers increasingly become a trend. This review presents several biomarkers of esophageal cancer, including chemotherapy-related biomarkers and targeted drug-related biomarkers, and the correlation of these biomarkers with drug response.

Detection of EGFR somatic mutations in non-small cell lung cancer (NSCLC) using a novel mutant-enriched liquidchip (MEL) technology.

We have developed and standardized a novel technology, mutant-enriched liquidchip (MEL), for clinical detection of EGFR mutations. The MEL integrates a mutant-enriched PCR procedure with liquidchip technology for detections of EGFR exon 19 deletions and L858R mutation on both formalin-fixed and paraffin-embedded (FFPE) slides and plasma samples from patients with non-small cell lung cancer (NSCLC). The detection sensitivity was 0.1% of mutant DNA in the presence of its wild-type DNA. The cross-reaction rate was lower than 5%. To evaluate the MEL platform, the EGFR mutation status of 59 patients with advanced NSCLC treated with EGFRTKIs (Tyrosine Kinase Inhibitors) were tested on their FFPE samples. EGFR exon 19 deletions and L858R were detected in 21 patients (21/59) and 76.2% (16/21) of them had partial response to the EGFR-TKIs, while by sequencing method, only 4 (4/59) mutations were detected. Plasma samples from 627 patients with various stages of NSCLC were examined with the MEL and 22% of EGFR exon 19 deletions and L858R were detected. Furthermore, in patients with advanced disease there are more mutations detected in plasma samples than in patients with less advanced disease. In conclusion, the MEL is a sensitive, stable, and robust technology for detecting EGFR DNA mutations from both FFPE and plasma samples from patients with NSCLC and is now routinely used for clinical diagnosis.

Gene expression significance in personalized medicine of non-small-cell lung cancer and gene expression analyzing platforms.

The association between gene expression and clinical characteristics of non-small cell lung cancer (NSCLC) are uncovered. These genes are critical elements in carcinoma physiological processes, including DNA synthesis, DNA repair and mitosis. Genes such as ERCC1, RRM1, TYMS, TUBB3 and STMN1 are predictive biomarker candidates for chemotherapy sensitivity in patients with NSCLC. Suitable gene expression analyzing technology is key factor for the personalize medicine to become a reality. This mini-review will describe and discuss critically on most currently widely used gene expression analyzing technologies, involving immunohistochemistry (IHC), reverse-transcription quantitative PCR (RT-qPCR) and branch-DNA technology (bDNA).