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// Xiaoyan Wang 1, 2 , Guanghua Chen 1, 2 , Chao Huang 1 , Hualei Tu 3 , Jilong Zou 4 and Jinglong Yan 1 1 Department of Orthopedics, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, P.R. China 2 Key Laboratory of the Ministry of Education, Ministry of Myocardial Ischemia, Harbin 150086, P.R. China 3 Burn Department, The Fifth Hospital of Harbin, Harbin 150086, P.R. China 4 Department of Orthopedics, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, P.R. China Correspondence to: Jinglong Yan, email: yanjinglong246@163.com Keywords: extracellular matrix, BMSCs, tissue engineering, scaffold, bone regeneration Received: August 24, 2017 Accepted: September 20, 2017 Published: October 09, 2017 ABSTRACT The extracellular matrix(ECM), which is primarily composed of collagens and proteoglycans, plays a key role in cell proliferation, differentiation, and migration and interactions between cells. In this study, we produced chitosan/gelatin/bone marrow stem cells-derived extracellular matrix(C/G/BMSCs-dECM) scaffolds via lyophilization and cross-linking, and chitosan/gelatin(C/G) scaffolds were used as controls. For the C/G/BMSCs-dECM scaffolds, the average pore size was 289.17 ± 80.28 μm; the average porosity was 89.25 ± 3.75%; the average compressive modulus was 0.82 ± 0.07 MPa; and the average water uptake ratio was 13.81 ± 1.00. In vitro , the C/G/BMSCs-dECM scaffolds promoted bone marrow stem cells(BMSCs) attachment and proliferation. Moreover, improved osteogenic differentiation was observed for these scaffolds. Thus, C/G/BMSCs-dECM is a promising material for bone tissue engineering.

Background/Aims: Osteosarcoma is the predominant form of primary bone malignancy. Although the combinational application of neoadjuvant chemotherapy and surgical resection significantly increases the survival rate, the therapeutic outcome remains unsatisfactory. Deoxyelephantopin (DET), an active ingredient of Elephantopus scaber, has been reported to have an anti-tumor effect in recent publications. This study aimed to investigate whether DET has antineoplastic effects on osteosarcoma cells and its underlying mechanism. Methods: Cell viability and morphological changes were assessed by MTT and Live/dead assays. Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential were detected utilizing Annexin V-FITC/PI double staining, DCFH-DA and JC-1 probes, respectively. Autophagy was detected by mRFP-GFP-LC3 adenovirus transfection and western blot. Results: DET dose-dependently reduced the viability of osteosarcoma cells following the increase in intracellular ROS levels. Pretreatment with N-acetylcysteine (NAC) reversed this effect. Furthermore, DET induced mitochondrial apoptosis. Depolarized cells were increased, and apoptosis-related proteins, such as Bax, Bcl-2, cleaved caspase-9, cleaved caspase-3 and cleaved ploy ADP-ribose polymerase, were activated. Additionally, we found that DET could induce autophagy in osteosarcoma cells, but autophagy inhibition did not affect the decrease in cell viability. Conclusion: DET induced apoptosis in osteosarcoma cells through ROS generation, mitochondrial dysfunction and caspase activation; in addition, autophagy was involved in the effects of DET on osteosarcoma cells.

Objective: To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on Staphylococcus aureus (SA) in vitro. Methods: hUCMSCs were isolated from umbilical cord tissues of full-term healthy fetuses after cesarean section and then cultured. Cells in the third passage were chosen for the use of experiment after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the following experiment. Cells were divided into 0, 10, 100 and 1,000 ng/ml lipopolysaccharide (LPS) groups by use of the random number table (similarly hereinafter). Cells were cultured with culture medium containing mesenchymal stem cells (MSCs) after being treated with medium containing corresponding mass concentrations of LPS for 12 h. At post culture hour (PCH) 6, 12 and 24, 6 wells of culture supernatant of cells in each group were obtained to measure the content of LL-37 with enzyme-linked immunosorbent assay (ELISA). Ninety blood agar culture plates were divided into ciprofloxacin control group (CC), ciprofloxacin + supernatant group (CS), and ciprofloxacin + supernatant + LL-37 antibody group (CSL), with 30 blood agar culture plates in each group. Blood agar culture plates in group CC were coated with 1.5 × 10 8 colony forming unit (CFU)/ml bacteria solution prepared with normal saline. Blood agar culture plates in group CS were coated with 1.5 × 10 8 CFU/ml bacteria solution prepared with normal saline and hUCMSC culture supernatant (cultured by MSC culture medium, the same below) in double volume of normal saline. Blood agar culture plates in group CSL were coated with 1.5 × 10 8 CFU/ml bacteria solution prepared with normal saline, hUCMSC culture supernatant in double volume of normal saline, and 2.6 μL of LL-37 antibodies at the concentration of 2 μg/ml. At PCH 12, 24 and 48, 10 blood agar culture plates were taken out from each group to observe the distribution of SA colonies on blood agar culture plates and to measure diameters of zones of inhibition of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA in each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in group CS and group CSL at PCH 12, 24 and 48 were calculated, with the synergistic effect evaluated. Data were processed with factorial design ANOVA, one way ANOVA, LSD- t test, Kruskal-Wallis test and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in cell culture supernatant in 10 ng/ml LPS group, 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 0 ng/ml LPS group (with t values ranging from 11.22 to 33.36, p values all below .01); the content of LL-37 in cell culture supernatant in either 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 10 ng/ml LPS group (with t values ranging from 2.24 to 18.73, p < .05 or p < .01); the content of LL-37 in cell culture supernatant in 1,000 ng/ml LPS group was higher than that in 100 ng/ml LPS group (with t values ranging from 12.46 to 14.70, p values all below .01). (2) At PCH 12, 24 and 48, the bacterial colonies in groups CC, CS and CSL became integrated over time. In CC group, diameters of zones of inhibition of ciprofloxacin at PCH 12, 24 and 48 were 26 mm, 24 mm and 23 mm respectively, with no obvious changes. At PCH 12, 24 and 48, diameters of zones of inhibition of ciprofloxacin in groups CS and CSL were 82 mm, 71 mm, 68 mm and 74 mm, 59 mm, 56 mm respectively, which were significantly larger than those in group CC. (3) At each PCH, MIC of ciprofloxacin against SA in group CC was significantly higher than that in groups CS and CSL respectively (with Z values ranging from 6.22 to 6.71, p values all below .01); MIC of ciprofloxacin against SA in group CSL was significantly higher than that in group CS (with Z values all equal to 6.72, p values all below .01). (4) FIC indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24 and 48 were 0.011, 0.032, 0.032 and 0.122, 0.350, 0.350, respectively. The results indicated that hUCMSC culture supernatant had a synergistically antibacterial effect when combined with ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is improved with increase of LPS concentration. The combination of hUCMSC culture supernatant with ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA effectively. Once LL-37 is neutralized, the synergistically antibacterial effect of hUCMSC culture supernatant is decreased thereby.