Regulation of osteogenic differentiation by the pro-inflammatory cytokines IL-1ß and TNF-α: current conclusions and controversies.

Treatment of complex bone fracture diseases is still a complicated problem that is urged to be solved in orthopedics. In bone tissue engineering, the use of mesenchymal stromal/stem cells (MSCs) for tissue repair brings hope to the medical field of bone diseases. MSCs can differentiate into osteoblasts and promote bone regeneration. An increasing number of studies show that the inflammatory microenvironment affects the osteogenic differentiation of MSCs. It is shown that TNF-α and IL-1ß play different roles in the osteogenic differentiation of MSCs via different signal pathways. The main factors that affect the role of TNF-α and IL-1ß in osteogenic differentiation of MSCs include concentration and the source of stem cells (different species and different tissues). This review in-depth analyzes the roles of pro-inflammatory cytokines in the osteogenic differentiation of MSCs and reveals some current controversies to provide a reference of comprehensively understanding.

The application of human periodontal ligament stem cells and biomimetic silk scaffold for in situ tendon regeneration.

BACKGROUND: With the development of tissue engineering, enhanced tendon regeneration could be achieved by exploiting suitable cell types and biomaterials. The accessibility, robust cell amplification ability, superior tendon differentiation potential, and immunomodulatory effects of human periodontal ligament stem cells (hPDLSCs) indicate their potential as ideal seed cells for tendon tissue engineering. Nevertheless, there are currently no reports of using PDLSCs as seed cells. Previous studies have confirmed the potential of silk scaffold for tendon tissue engineering. However, the biomimetic silk scaffold with tendon extracellular matrix (ECM)-like structure has not been systematically studied for in situ tendon regeneration. Therefore, this study aims to evaluate the effects of hPDLSCs and biomimetic silk scaffold on in situ tendon regeneration. METHODS: Human PDLSCs were isolated from extracted wisdom teeth. The differentiation potential of hPDLSCs towards osteo-, chondro-, and adipo-lineage was examined by cultured in different inducing media. Aligned and random silk scaffolds were fabricated by the controlled directional freezing technique. Scaffolds were characterized including surface structure, water contact angle, swelling ratio, degradation speed and mechanical properties. The biocompatibility of silk scaffolds was evaluated by live/dead staining, SEM observation, cell proliferation determination and immunofluorescent staining of deposited collagen type I. Subsequently, hPDLSCs were seeded on the aligned silk scaffold and transplanted into the ruptured rat Achilles tendon. Scaffolds without cells served as control groups. After 4 weeks, histology evaluation was carried out and macrophage polarization was examined to check the repair effects and immunomodulatory effects. RESULTS: Human PDLSCs were successfully isolated, and their multi-differentiation potential was confirmed. Compared with random scaffold, aligned silk scaffold had more elongated and aligned pores and promoted the proliferation and ordered arrangement of hPDLSCs. After implantation into rat Achilles tendon defect, hPDLSCs seeded aligned silk scaffold enhanced tendon repair with more tendon-like tissue formation after 4 weeks, as compared to the scaffold-only groups. Higher expression of CD206 and lower expression of iNOS, IL-1ß and TNF-α were found in the hPDLSCs seeded aligned silk scaffold group, which revealed its modulation effect of macrophage polarization from M1 to M2 phenotype. CONCLUSIONS: In summary, this study demonstrates the efficacy of hPDLSCs as seed cells and aligned silk scaffold as a tendon-mimetic scaffold for enhanced tendon tissue engineering, which may have broad implications for future tendon tissue engineering and regenerative medicine researches.

[Antitumor Activity of TRAIL-Mu3 Protein in vitro and in vivo and the Mechanisms].

OBJECTIVE: TRAIL-Mu3 was obtained by mutating the N-terminus of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene to an eight continuous arginine sequence. The present study was designed to explore the antitumor effect of this soluble mutant protein and the underlying mechanisms. METHODS: The inhibitory effect of TRAIL-Mu3 on the proliferation of lung cancer cell lines NCI-H460, A549, NCI-H1299 and calu-1 was tested by CCK8 assay. The apoptotic rates of A549 and NCI-H460 treated by TRAIL-Mu3 were detected by flow cytometer (FCM). The expressions of apoptosis related proteins death receptor (DR) 4, DR5, Caspase-3, Caspase-8 and X-linked inhibitor of apoptosis protein (XIAP) were detected by Western blot .Moreover, a subcutaneous xenograft tumor mouse model of NCI-H460 was established and treated with TRAIL-Mu3 daily or every other day or three times a week. The expressions of DR4, DR5, Caspase-3, Caspase-8 and XIAP were detected by immunohistochemical staining. RESULTS: The in vitro study demonstrated that as compared to the TRAIL, the TRAIL-Mu3 was more toxic and pro-apoptotic by up-regulation of the expression and activity of DR4, Caspase-3 and Caspase-8. Also, the animal study showed a similar antitumor effect between treatment with TRAIL-Mu3 every other day and three time a week, which was better than daily use. All treatments significantly suppressed the growth of xenograft tumor, increased the expression or activity of DR4 and Caspase-3, and down-regulated the expression of XIAP ( P<0.05). CONCLUSION: TRAIL-Mu3 could improve antitumor activity in vivo and in vitro through elevating DR4 expression, activating Caspase-3/-8, and inhibiting XIAP activation.

Generation of a novel TRAIL mutant by proline to arginine substitution based on codon bias and its antitumor effects.

TNF ligand superfamily member 10 (TRAIL) is a member of the tumor necrosis factor superfamily. The present study was performed in an effort to increase the expression of soluble (s)TRAIL by rebuilding the gene sequence of TRAIL. Three principles based on the codon bias of Escherichia coli were put forward to design the rebuild strategy. Relying on these three principles, a P7R mutation near the N­terminal region of sTRAIL, named TRAIL­Mu, was designed. TRAIL­Mu was subsequently cloned into the PTWIN1 plasmid and expressed in E. coli BL21 (DE3). Using a high­level expression system and a three­step purification method, soluble TRAIL­Mu protein reached ~90% of total cellular protein and purity was >95%, demonstrating success in overcoming inclusion body formation. The cytotoxic effect of TRAIL­Mu was evaluated by sulforhodamine B assay in the MD­MB­231, A549, NCI­H460 and L02 cell lines. The results demonstrated that TRAIL­Mu exerted stronger antitumor effects on TRAIL­sensitive tumor cell lines, and was able to partially reverse the resistance of a TRAIL­resistant tumor cell line. In addition, TRAIL­Mu exhibited no notable biological effects in a normal liver cell line. The novel TRAIL variant generated in the present study may be useful for the mass production of this important protein for therapeutic purposes.

TRAIL mutant membrane penetrating peptide alike-MuR6-TR enhances the antitumor effects of TRAIL in pancreatic carcinoma both in vitro and in vivo.

To remedy the drug resistance of natural tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and enhance its antitumor effects, we prepared a type of TRAIL mutant membrane penetrating peptide alike (TMPPA)­TRAIL mutant R6 (MuR6-TR) by mutating the N­terminal of the soluble TRAIL gene sequence. The expressed MuR6­TR protein was purified to treat pancreatic carcinoma cell lines BxPC­3 and PANC­1. The inhibitory effects on the proliferation of BxPC­3 and PANC­1 cells was assessed with CCK­8 assay and compared with natural TRAIL. The antitumor effect of MuR6­TR was assessed on implant tumors derived from PANC­1 cells in nude mice and compared with gemcitabine. Finally, the soluble MuR6­TR gene was successfully mutated with 4 amino acids in the N­terminal of TRAIL and had a molecular size of 513 bp. The mutant MuR6­TR was connected to pET32a and verified by enzymatic digestion and sequencing. The recombinant MuR6­TR was transformed and expressed in Escherichia coli. The CCK­8 assay results indicated that MuR6­TR inhibited the growth of BxPC­3 and PANC­1 cells in a dose­dependent manner, with IC50 values of 4.63 and 7.84 ng/ml, respectively, which were much lower than that of natural TRAIL. MuR6­TR demonstrated a higher inhibitory effect on tumor growth (24.2%) than natural TRAIL (14.4%) and an effect similar to that of gemcitabine at an early period. Thus, the mutant MuR6­TR exhibited a stronger antitumor effect than that of natural TRAIL both in vivo and in vitro and may have potential therapeutic value for pancreatic carcinoma, which requires further validation.

Exogenous jasmonic acid can enhance tolerance of wheat seedlings to salt stress.

Jasmonic acid (JA) is regarded as endogenous regulator that plays an important role in regulating stress responses, plant growth and development. To investigate the physiological mechanisms of salt stress mitigated by exogenous JA, foliar application of 2mM JA was done to wheat seedlings for 3days and then they were subjected to 150mM NaCl. Our results showed that 150mM NaCl treatment significantly decreased plant height, root length, shoot dry weight, root dry weight, the concentration of glutathione (GSH), chlorophyll b (Chl b) and carotenoid (Car), the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), enhanced the concentration of malondialdehyde (MDA), hydrogen peroxide (H2O2) and the rate of superoxide radical (O2•-) generation in the wheat seedlings when compared with the control. However, treatments with exogenous JA for 3 days significantly enhanced salt stress tolerance in wheat seedlings by decreasing the concentration of MDA and H2O2, the production rate of O2•- and increasing the transcript levels and activities of SOD, POD, CAT and APX and the contents of GSH, Chl b and Car, which, in turn, enhanced the growth of salt stressed seedlings. These results suggested that JA could effectively protect wheat seedlings from salt stress damage by enhancing activities of antioxidant enzymes and the concentration of antioxidative compounds to quench the excessive reactive oxygen species caused by salt stress and presented a practical implication for wheat cultivation in salt-affected soils.