3D Printed Platelet-Rich Plasma-Loaded Scaffold with Sustained Cytokine Release for Bone Defect Repair.

The combination of three-dimensional (3D) printed scaffold materials and various cytokines can achieve the purpose of tissue reconstruction more efficiently. In this study, we prepared platelet-rich plasma (PRP)/gelatin microspheres combined with 3D printed polycaprolactone/ß-tricalcium phosphate scaffolds to solve the key problem that PRP cannot be released under control and the release time is too short, and thus better promote bone repair. Consequently, the composite scaffold displayed a good mechanical property and sustained cytokine release for ∼3 weeks. Increased survival, proliferation, migration, and osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells were observed compared with the control groups. The in vivo study demonstrated that the composite scaffold with PRP/gelatin microspheres led to greater positive effects in promoting large bone defect repair. In conclusion, in this study, a new type of PRP long-term sustained-release composite scaffold material was constructed that effectively improved the survival, proliferation, and differentiation of cells in the transplanted area, thereby better promoting the repair of large bone defects. Impact statement Reconstruction of bone tissue and blood vessels at bone defects takes time. Platelet-rich plasma (PRP) has been widely used in bone defect repair because it contains a variety of cytokine that can promote local osteogenesis and angiogenesis. In this study, we constructed a new type of polycaprolactone/ß-tricalcium phosphate/PRP/gelatin scaffold to solve the predicament of short cytokine release time in PRP-related materials. We proved that this scaffold can not only achieve long-term PRP-related cytokine release (more than 3 weeks) but also promote osteogenesis and bone defect repair. We believe that this is a novel concept of developing the sustained PRP-related cytokine releasing bioscaffold for treating large bone defect.

3D printed polycaprolactone/beta-tricalcium phosphate/magnesium peroxide oxygen releasing scaffold enhances osteogenesis and implanted BMSCs survival in repairing the large bone defect.

Ischemia and hypoxia in the bone defect area remain an intractable problem when treating large bone defects. Thus, oxygen-releasing biomaterials have been widely researched in recent years. Magnesium peroxide (MgO2) can release oxygen (O2), and magnesium ions (Mg2+), simultaneously, which is seen to have significant potential in bone substitutes. In this study, we used 3D printing technology to fabricate a MgO2-contained composite scaffold, which was composed of polycaprolactone (PCL), beta-tricalcium phosphate (ß-TCP) and magnesium peroxide (MgO2). Physical properties and O2/Mg2+ releasing behavior of the scaffold were studied. Then, we evaluated the effects of the scaffold on cell survival, proliferation, migration, adhesion and osteogenic differentiation by the co-culture of bone marrow mesenchymal stem cells (BMSCs) and scaffold under normoxia and hypoxia in vitro. Finally, the osteogenic properties of the scaffold in vivo were evaluated via the rat femoral condylar bone defect model. The PCL/ß-TCP/MgO2 scaffold showed good mechanical properties and sustained O2 and Mg2+ release for about three weeks. Meanwhile, the scaffold showed appreciable promotion on the survival, proliferation, migration and osteogenic differentiation of BMSCs under hypoxia compared with control groups. The results of imaging studies and histological analysis showed that implantation of PCL/ß-TCP/MgO2 scaffold could promote seed cell survival and significantly increased new bone formation. In sum, the PCL/ß-TCP/MgO2 scaffold is promising with great potential for treating large bone defects.

The Regulatory Effect of VEGF-Ax on Rat Bone Marrow Mesenchymal Stem Cells' Angioblastic Differentiation and Its Proangiogenic Ability.

Vascular endothelial growth factor A (VEGFA), which plays a key role in angiogenesis, is composed of many isoforms. Distinct VEGFA isoforms are generated by alternative splicing of VEGFA mRNA and named as VEGFxxx, where xxx represents the number of amino acids present in the final protein sequence. These isoforms have opponent pro- and antiangiogenic effects. VEGF-Ax, an additional isoform containing a 22-amino-acid extension in the COOH terminus, arising from VEGFA mRNA, programmed translational readthrough. The function of VEGF-Ax is not clear, especially the conclusion that VEGF-Ax regulates angiogenesis is contradictory. Thus, we investigated the effect of VEGF-Ax on differentiation and angiogenesis of rat bone marrow mesenchymal stem cells (BMMSCs). The results showed that VEGF-Ax could promote the proliferation and migration of BMMSCs, stimulate the differentiation of BMMSCs into endothelial cell-like cells, and protect BMMSCs from endoplasmic reticulum stress-induced apoptosis.

Downregulation of SAFB Sustains the NF-κB Pathway by Targeting TAK1 during the Progression of Colorectal Cancer.

Purpose: To investigate the role and the underlying mechanism of scaffold attachment factor B (SAFB) in the progression of colorectal cancer (CRC).Experimental Design: SAFB expression was analyzed in the Cancer Outlier Profile Analysis of Oncomine and in 175 paraffin-embedded archived CRC tissues. Gene Ontology analyses were performed to explore the mechanism of SAFB in CRC progression. Western blot, RT-PCR, luciferase assay, and chromatin immunoprecipitation (ChIP) were used to detect the regulation of transforming growth factor-ß-activated kinase 1 (TAK1) and NF-κB signaling by SAFB The role of SAFB in invasion, metastasis, and angiogenesis was investigated using in vitro and in vivo assays. The relationship between SAFB and TAK1 was analyzed in CRC tissues.Results: SAFB was downregulated in CRC tissues, and low expression of SAFB was significantly associated with an aggressive phenotype and poorer survival of CRC patients. The downregulation of SAFB activated NF-κB signaling by targeting the TAK1 promoter. Ectopic expression of SAFB inhibited the development of aggressive features and metastasis of CRC cells both in vitro and in vivo The overexpression of TAK1 could rescue the aggressive features in SAFB-overexpressed cells. Furthermore, the expression of SAFB in CRC tissues was negatively correlated with the expression of TAK1- and NF-κB-related genes.Conclusions: Our results show that SAFB regulated the activity of NF-κB signaling in CRC by targeting TAK1 This novel mechanism provides a comprehensive understanding of both SAFB and the NF-κB signaling pathway in the progression of CRC and indicates that the SAFB-TAK1-NF-κB axis is a potential target for early therapeutic intervention in CRC progression. Clin Cancer Res; 23(22); 7108-18. ©2017 AACR.

TLE3 represses colorectal cancer proliferation by inhibiting MAPK and AKT signaling pathways.

BACKGROUND: Transducin-like enhancer of Split3 (TLE3) serves as a transcriptional corepressor during cell differentiation and shows multiple roles in different kinds of cancers. Recently, TLE3 together with many other genes involved in Wnt/ß-catenin pathway were detected hyper-methylated in colorectal cancer (CRC). However, the potential role and the underlying mechanism of TLE3 in CRC progression remain scarce. METHODS: Gene expression profiles were analyzed in The Cancer Genome Atlas (TCGA) microarray dataset of 41 normal colorectal intestine tissues and 465 CRC tissues. Western blot and Real-time Quantitative PCR (RT-qPCR) were respectively performed to detect protein and mRNA expression in 8 pairs of CRC tissue and matched adjacent normal mucosa. Immunohistochemistry (IHC) was conducted to evaluate TLE3 protein expression in 105 paraffin-embedded, archived human CRC tissues from patients, whose survival data were analyzed with Kaplan-Meier method. In vitro experiments including MTT assay, colony formation assay, and soft agar formation assay were used to investigate the effects of TLE3 on CRC cell growth and proliferation. Additionally, subcutaneous tumorigenesis assay was performed in nude mice to confirm the effects of TLE3 in vivo. Furthermore, gene set enrichment analysis (GSEA) was run to explore potential mechanism of TLE3 in CRC, and then we measured the distribution of CRC cell cycle phases and apoptosis by flow cytometry, as well as the impacts of TLE3 on MAPK and AKT signaling pathways by Western blot and RT-qPCR. RESULTS: TLE3 was significantly down-regulated in 465 CRC tissues compared with 41 normal tissues. Both protein and mRNA expressions of TLE3 were down-regulated in CRC compared with matched adjacent normal mucosa. Lower expression of TLE3 was significantly associated with poorer survival of patients with CRC. Besides, knock down of TLE3 promoted CRC cell growth and proliferation, while overexpression of TLE3 showed suppressive effects. Furthermore, overexpression of TLE3 caused G1-S phase transition arrest, inhibition of MAPK and AKT pathways, and up-regulation of p21Cip1/WAF1 and p27Kip1. CONCLUSION: This study indicated that TLE3 repressed CRC proliferation partly through inhibition of MAPK and AKT signaling pathways, suggesting the possibility of TLE3 as a biomarker for CRC prognosis.