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Objective To prepare a bionic artificial bone scaffold using a room temperature three dimensional (3D) printing technique and evaluate its biocompatibility and bioactivity in vitro.Methods A room temperature 3D printing technique was applied to fabricate 3D bionic artificial bone scaffolds using collagen/hydroxyapatite.The physico-chemical structure,porosity and mechanical strength of the scaffolds were assessed.The extract liquid of scaffolds was cocultured with bone mesenchymal stem cells (BMSCs) to evaluate the toxicity of scaffolds.There were 3 experimental groups:blank control with no scaffolds,printed scaffolds group and non-printed scaffolds group.The condition of BMSCs on the scaffolds was observed via scanning electron microscopy(SEM) and immunostaining.3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and SEM were applied to monitor the proliferation of BMSCs on the scaffolds.At last,alkaline phosphatase (ALP) activity and mRNA expression levels of osteogenesis-related genes were detected to assess the osteoinductive property of the scaffolds.Results The 3D printed scaffolds fabricated in the present study were characterized by highly interconnected pores which were controllable and even in size.The cross section of the scaffolds presented an irregular honeycomb-like microstructure.The porosity of printed 3D scaffolds (71.14％ ± 2.24％) was significantly higher than that of non-printed scaffolds (59.04％ ±2.98％) (P ＜ 0.05).The physico-chemical structures of the materials were preserved after printing without additional cytotoxicity.The MTT results at 7 and 14 days revealed that the printed scaffolds had a significantly more cell numbers than the non-printed scaffolds(P ＜ 0.05).SEM showed that the BMSCs adhered well onto the printed scaffolds and proliferated and migrated through the pores.Compared with the blank control,the printed scaffolds showed obviously better osteogenic outcomes.Conclusion The 3D bionic artificial bone scaffolds of collagen/hydroxyapatite manufactured by a room temperature 3D printing technique can provide a good extracellular matrix for BMSCs to proliferate and differentiate.
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Objective To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) transplantation on expression of high mobility group box 1 protein (HMGB1) in rats with acute liver failure (ALF).Method SD rats were randomly divided into three groups:control group,ALF group and BMSCs transplantation group.Specimens were harvested in each group at 12 h,24 h and 72 h after ALF induction.Serum ALT and AST concentrations were determined,liver pathological changes were observed by HE staining,serum HMGB1 concentration detected by ELISA,and HMGB1 mRNA and protein in liver tissues examined by RT-PCR and immunohistochemistry respectively.Result The ALF models were successfully induced by D-GalN (900 rng/kg) and LPS (10 μg/kg) injection.Serum levels of ALT and AST in the ALF group were gradually increased with progression of the disease.As compared with the ALF group,significant improvement of liver function parameters and histological findings was observed in the transplantation group 72 h after transplantation (P＜0.01).The serum HMGB1 concentrations,the HMGB1 mRNA expression and the HMGB1 protein expression in liver tissue of control group were lowered at all time points,and they increased with time in the ALF group.After BMSCs transplantation,the serum HMGB1 concentrations,the HMGB1 mRNA and protein expression decreased with time.All the differences between ALF group and BMSCs transplantation group at 24 h and 72 h after transplantation were statistically significant (P＜0.01).At 24th h after transplantation,mortality rates of control group,ALF group,BMSC transplantation group were 0 (0/24),33.3％ (8/24) and 16.7％ (4/24) respectively with the difference being statistically significant (x2 =21.098,P＜ 0.01).Conclusion BMSCs transplantation can improve the liver function and pathological changes in rats with ALF,and decrease the expression of HMGB1.
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Objective To investigate the in vitro cytocompatibility of three-dimensional porous scaffolds of poly-D,L-lactic acid (PDLLA) and discuss the feasibility of PDLLA as a scaffold for bone tissue engineering.Methods BMSCs of the third passage were seeded on osteogenetic differentiation medium or culture medium containing 20％ volume fraction degraded liquid (PDLLA degradation liquid of 0,3,6,9,and 12 weeks) according to the random number table.Osteogenetic differentiation medium or culture medium without PDLLA was used as controls.Cell viability,cytotoxicity,and osteogenic differentiation were detected for study on cytocompatibility of PDLLA.Scanning electron microscopy was used to observe the growth of BMSCs on the surface of PDLLA scaffolds.Results PDLLA scaffolds presented no significant cytotoxic on the growth of BMSCs.PDLLA scaffolds had no negative effect on cell viability compared with the controls (t3 =-0.441,P =0.671; t6 =1.596,P =0.154; t9 =-0.492,P =0.636; t12 =-1.135,P=0.283).ALP staining and calcium nodule staining were positive and there were no significant differences in ALP and collagen Ⅰ protein quantitative detection compared with the controls.BMSCs grew well on the inner surface of the PDLLA three-dimensional porous scaffolds.Conclusion Three-dimensional porous scaffolds of PDLLA present good cytocompatibility in vitro and can be used as bone tissue engineering scaffolds for subsequent in vivo research.
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Objective To induce the immune tolerance of heart grafts with infusion of isogeneic bone marrow mesenchymal stem cells (BMSCs) in heart transplant rats.Method Donor Wistar rats and recipient F344 rats were randomly divided into 4 groups:acute rejection group (group A),Wistar rats as the donors and F344 rats as the recipients for heart transplantation; low dose cyclosporin A(CsA) group (group B),recipient F344 rats given low dose CsA; BMSCs group (group C),recipient F344 rats given isogeneic BMSCs; BMSC and low dose CsA group (group D),the recipient F344 rats given isogeneic BMSCs and low dose CsA.The serum cytokine levels were determined,and the donor heart pathological changes and survival were observed postoperatively.The relative level of Foxp3 mRNA expression in the spleen of the recipient F344 rats was also observed.Result The blood levels of interleukin-2 (IL-2) and interferon-γ(INF-γ) were significantly reduced,but IL-4 and IL-10 levels were increased (P＜0.05),and the survival time of donor heart was significantly prolonged in group D as compared with groups A,B and C (P＜0.05 for all).Heart pathological examination revealed a mild acute rejection in group D,moderate acute rejection in groups B and C group,and severe acute rejection in group A respectively.The expression of Foxp3 mRNA was significantly lower in group A than in groups B,C and D (P＜0.05 for all),and that in group D was significantly higher than in groups B and C (P＜0.05 for both),but there was no significant difference between between groups B and C (P＞0.05).Conclusion Intravenous administration of BMSCs can alleviate immunorejection in heterotopic rat heart transplantation.Low-dose CsA acts synergistically with BMSCs to significantly inhibit acute rejection after heart transplantation.The partial mechanisms involve the suppressive effect of BMSCs on the expression of Foxp3 mRNA and modulation on cytokine.
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Mesenchymal stem cells (MSCs) are promising candidates to develop new cell-based therapeutic strategies and bone marrow represents the main source of MSCs for experimental and clinical application currently. How to obtain enough MSCs is the biggest challenge faced by the experimental and clinical application. The difference of morphology, growth pattern, immunophenotype and multilineage differentiation of MSCs derived from human umbilical cord vein and their multiple differentiation capacity were investigated. The results showed that MSCs derived from human umbilical cord vein and normal adult bone marrow were similar in biological characteristics. They not only grew in fibroblast-like cells mode but had great expansion potency and multilineage differentiation ability in vitro. The MSCs derived from human umbilical cord vein may be an excellent alternative source for experimental and clinical application.