Daping Wang

Telomerase Reverse Transcriptase Gene Promoter Mutations Help Discern the Origin of Urogenital Tumors: A Genomic and Molecular Study

Activation of telomerase can be observed in almost all human tumor histotypes and detection of the urinary telomerase activities is useful for the diagnosis and surveillance of bladder cancer. In this study, we screened, by Sanger sequencing, 302 patients with various urogenital cancers for somatic mutations in the promoter of the telomerase reverse transcriptase (TERT) gene and determined the clinical relevance of TERT promoter mutations in urogenital cancer. In vitro assays were also performed to evaluate the functional influence of the discovered mutations. We found that the frequencies of somatic mutations in the TERT promoter varied substantially between different types of urogenital tumors (range: 0-63.7%), with urothelial carcinomas showing the highest mutation frequency and prostate cancer showing no mutation. The mutations upregulated the expression of TERT and enhanced the invasiveness of the tumor cells. The mutations were more prevalent in older patients with invasive diseases and advanced tumor stages, and were associated with significantly shorter survival time. Moreover, we also observed a significant co-occurrence of mutations between the TERT promoter and the tumor protein 51/retinoblastoma1 (TP53/RB1) signaling pathway. Hence, TERT promoter mutations may serve as important markers for the differential diagnosis and surveillance of urogenital tumors.

Extracellular matrix production in vitro in cartilage tissue engineering

Cartilage tissue engineering is arising as a technique for the repair of cartilage lesions in clinical applications. However, fibrocartilage formation weakened the mechanical functions of the articular, which compromises the clinical outcomes. Due to the low proliferation ability, dedifferentiation property and low production of cartilage-specific extracellular matrix (ECM) of the chondrocytes, the cartilage synthesis in vitro has been one of the major limitations for obtaining high-quality engineered cartilage constructs. This review discusses cells, biomaterial scaffolds and stimulating factors that can facilitate the cartilage-specific ECM production and accumulation in the in vitro culture system. Special emphasis has been put on the factors that affect the production of ECM macromolecules such as collagen type II and proteoglycans in the review, aiming at providing new strategies to improve the quality of tissue-engineered cartilage.

Multilayered molecular profiling supported the monoclonal origin of metastatic renal cell carcinoma

Primary renal cell carcinomas (pRCCs) have a high degree of intratumoral heterogeneity and are composed of multiple distinct subclones. However, it remains largely unknown that whether metastatic renal cell carcinomas (mRCCs) also have startling intratumoral heterogeneity or whether development of mRCCs is due to early dissemination or late diagnosis. To decipher the evolution of mRCC, we analyzed the multilayered molecular profiles of pRCC, local invasion of the vena cava (IVC), and distant metastasis to the brain (MB) from the same patient using whole‐genome sequencing, whole‐exome sequencing, DNA methylome profiling, and transcriptome sequencing. We found that mRCC had a lower degree of heterogeneity than pRCC and was likely to result from recent clonal expansion of a rare, advantageous subclone. Consequently, some key pathways that are targeted by clinically available drugs showed distinct expression patterns between pRCC and mRCC. From the genetic distances between different tumor subclones, we estimated that the progeny subclone giving rise to distant metastasis took over half a decade to acquire the full potential of metastasis since the birth of the subclone that evolved into IVC. Our evidence supported that mRCC was monoclonal and distant metastasis occurred late during renal cancer progression. Thus, there was a broad window for early detection of circulating tumor cells and future targeted treatments for patients with mRCCs should rely on the molecular profiles of metastases.

Evaluation of the novel three-dimensional porous poly (L-lactic acid)/nano-hydroxyapatite composite scaffold.

To determine the optimal ratio of nano-hydroxyapatite (n-HA) to polylactic acid (PLLA) in the novel three-dimensional porous PLLA/n-HA composite scaffolds, low-temperature rapid prototyping technology was employed to fabricate the composite materials with different n-HA contents. Mechanical properties and degradation behaviors of the composites were examined, and the scaffold microstructure and n-HA dispersion were observed by scanning electron microscope (SEM). Mechanical tests demonstrated that the tensile strength of the composite material gradually decreased with an increase in n-HA content. When the n-HA content reached 20 wt%, the bending strength of the composite material peaked at 138.5 MPa. SEM images demonstrated that the optimal content of n-HA was 20 wt% as the largest interconnected pore size that can be seen, with a porosity as high as 80%. In vitro degradation experiments demonstrated that the pH value of the material containing solution gradually decreased in a time-dependent manner, with a simultaneous weakening of the mechanical properties. In vitro study using rat osteoblast cells showed that the composite scaffolds were biocompatible; the 20 wt% n-HA scaffold offered particular improvement to rat osteoblast cell adhesion and proliferation compared to other compositions. It was therefore concluded that 20 wt% n-HA is the optimal nano-hydroxyapatite (n-HA) to polylactic acid (PLLA) ratio, with promise for bone tissue engineering.

Effect of inorganic/organic ratio and chemical coupling on the performance of porous silica/chitosan hybrid scaffolds

Inorganic/organic hybrid scaffolds have great potential for tissue engineering applications due to controllable mechanical properties and tailorable biodegradation. Here, silica/chitosan hybrid scaffolds were fabricated through the sol-gel method with a freeze drying process. 3-Glycidoxypropyl trimethoxysilane (GPTMS) and tetraethylorthosilicate (TEOS) were used as the covalent inorganic/organic coupling agent and the separate inorganic source, respectively. Hybrid scaffolds with various inorganic/organic weight ratios (I/Os) and molar ratios of chitosan and GPTMS (GCs) were examined and compared in this study. FTIR showed that higher GPTMS content resulted in the increased covalent cross-linking of the chitosan and the silica network in hybrids. Compression testing indicated that increasing the GPTMS content greatly improved the compressive strength of scaffold. LIVE/DEAD assay showed that enhanced cytocompatibility was obtained as the silica content increased. Therefore, the results confirmed that the two parameters I/O and GC can largely influence the scaffold performance, which can be used to tailor the hybrid properties.

Low-temperature deposition manufacturing: A novel and promising rapid prototyping technology for the fabrication of tissue-engineered scaffold

Developed in recent years, low-temperature deposition manufacturing (LDM) represents one of the most promising rapid prototyping technologies. It is not only based on rapid deposition manufacturing process but also combined with phase separation process. Besides the controlled macropore size, tissue-engineered scaffold fabricated by LDM has inter-connected micropores in the deposited lines. More importantly, it is a green manufacturing process that involves non-heating liquefying of materials. It has been employed to fabricate tissue-engineered scaffolds for bone, cartilage, blood vessel and nerve tissue regenerations. It is a promising technology in the fabrication of tissue-engineered scaffold similar to ideal scaffold and the design of complex organs. In the current paper, this novel LDM technology is introduced, and its control parameters, biomedical applications and challenges are included and discussed as well.

The Study on Biocompatibility of Porous nHA/PLGA Composite Scaffolds for Tissue Engineering with Rabbit Chondrocytes In Vitro

Objective. To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA) composite and evaluate its feasibility as a scaffold for cartilage tissue engineering. Methods. Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffold in vitro and the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM). The cell cycle profile was analyzed by flow cytometry. Results. The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P > 0.05). Conclusion. The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs) and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2) and decreased type I collagen (COL1) protein expression levels. SRY-box 9 (SOX9) mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

Osteogenic differentiation of bone marrow mesenchymal stem cells by magnetic nanoparticle composite scaffolds under a pulsed electromagnetic field

This study was conducted to investigate the effect of magnetic nanoparticle composite scaffold under a pulsed electromagnetic field on bone marrow mesenchymal stem cells (BMSCs), which was achieved by examining the biological behaviors of cell adhesion, proliferation and differentiation on the surface of the scaffolds. This may provide some experimental evidence for the use of magnetic nanoparticles in medical application. The magnetic nanoparticle composite scaffolds were evaluated and characterized by the following indexes: the cell proliferation was detected by the CCK-8 method, the alkaline phosphatase (ALP) activity was examined by a detection kit, and the expression of type I collagen and osteocalcin gene were evaluated by RT-PCR. The CCK-8 test showed that there was no significant difference in Group A (BMSCs-seeded magnetic scaffolds under the electromagnetic field), B (BMSCs-seeded magnetic scaffolds) and C (BMSCs cultured alone) (P > 0.05). The value for the ALP activity in Group A was higher than the other two groups. In addition, the RT-PCR results showed that the expression of type I collagen gene in Group A was enhanced (P < 0.05), suggesting that the magnetic nanoparticles combined with the pulsed electromagnetic field had a positive effect on the osteogenic differentiation of BMSCs. However, the expression of osteocalcin was not significantly different in three groups (P > 0.05). To conclude, magnetic nanoparticles may induce the osteogenic differentiation with the action of the pulsed electromagnetic field.

Identification of a novel EXT1 mutation in patients with hereditary multiple exostosis by exome sequencing

Hereditary multiple exostosis (HME) is an autosomal inherited skeletal disease whose etiology is not fully understood. To further understand the genetic spectrum of the disease, we analyzed a five-generation Chinese family with HME that have observable inheritance. Exome sequencing was performed on three HME individuals and three unaffected individuals from the family. A downstream study confirmed a new C deletion at codon 442 on exon 5 of the exostosin-1 (EXT1) gene as the only pathogenic site which generated a stop codon and caused the truncation of the protein. We rediscovered the deletion in other affected individuals but not in the unaffected individuals from the family. Upon immunohistochemistry assay, we found that the EXT1 protein level of the patients with the novel mutation in our study was less than the level in the patients without the EXT1 mutation from another unrelated family. For a deeper understanding, we analyzed the mutation spectrum of the EXT1 gene. The present study should facilitate a further understanding of HME.