Objective To research in vitro biocompatibility of silicon containing micro-arc oxidation (MAO) coated magnesium alloy ZK60 with osteoblasts. Methods The surface microstructure of silicon containing MAO coated magnesium alloy ZK60 was observed by a scanning electron microscopy (SEM), and chemical composition of the coating surface was determined by energy dispersive spectrum analysis. The experiments were divided into 4 groups: silicon containing MAO coated magnesium alloy ZK60 group (group A), uncoated magnesium alloy ZK60 group (group B), titanium alloy group (group C), and negative control group (group D). Extracts were prepared respectively with the surface area to extraction medium ratio (1.25 cm2/ mL) according to ISO 10993-12 standard in groups A, B, and C, and were used to culture osteoblasts MC3T3-E1. The α-MEM medium supplemented with 10% fetal bovine serum was used as negative control in group D. The cell morphology was observed by inverted phase contrast microscopy. MTT assay was used to determine the cell viability. The activity of alkaline phosphatase (ALP) was detected. Cell attachment morphology on the surface of different samples was observed by SEM. The capability of protein adsorption of the coating surface was assayed, then DAPI and calcein-AM/ethidium homodimer 1 (calcein-AM/EthD-1) staining were carried out to observe cell adhesion and growth status. Results The surface characterization showed a rough and porous layer with major composition of Mg, O, and Si on the surface of silicon containing MAO coated magnesium alloy ZK60 by SEM. After cultured with the extract, cells grew well and presented good shape in all groups by inverted phase contrast microscopy, group A was even better than the other groups. At 5 days, MTT assay showed that group A presented a higher cell proliferation than the other groups (P lt; 0.05). Osteoblasts in groups A and C presented a better cell extension than group B under SEM, and group A exhibited better cell adhesion and affinity. Protein adsorption in group A [ (152.7 ± 6.3) µg/mL] was significantly higher than that of group B [(96.3 ± 3.9) µg/mL] and group C [ (96.1 ± 8.7) µg/mL] (P lt; 0.05). At each time point, the adherent cells on the sample surface of group A were significantly more than those of groups B and C (P lt; 0.05). The calcein-AM/EthD-1 staining showed that groups A and C presented better cell adhesion and growth status than group B. The ALP activities in groups A and B were 15.55 ± 0.29 and 13.75 ± 0.44 respectively, which were significantly higher than those in group C (10.43 ± 0.79) and group D (10.73 ± 0.47) (P lt; 0.05), and group A was significantly higher than group B (P lt; 0.05). Conclusion The silicon containing MAO coated magnesium alloy ZK60 has obvious promoting effects on the proliferation, adhesion, and differentiation of osteoblasts, showing a good biocompatibility, so it might be an ideal surface modification method on magnesium alloys.
Objective To summary the functional roles and molecular mechanisms of microRNA (miRNA) in osteoblast differentiation so as to supply information for basic and cl inical researches. Methods Recent l iterature concerning miRNA in osteoblast differentiation was reviewed. The information was classified and summarized. Results miRNAs critically regulate bone morphogenetic protein, transforming growth factor β, and Wnt/β-catenin signal ing pathways during osteoblast differentiation. In pathological conditions, especially in some disorders of abnormal osteoblast differentiation, downregulated miRNA expression has been observed. Conclusion miRNA may represent a novel biomarker for diagnosis, and a candidate target therapies for the disorders with abnormal osteoblast differentiation.
Objective To study the effect of signal-selective parathyroid hormone (PTH) analogue peptide on Wnt signal ing factors in osteoblasts isolated from neonatal mouse, and provide theoretical basis for the mechanism of PTH’s function in bone metabolism. Methods Osteoblasts were isolated from calvaria of 2-3-day-old C57BL neonatal mouse and identified by alkal ine phosphatase (ALP) staining, and Alizarin red staining. The cells at passage 1 were divided into 4 groups: control group, PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group. Then the medium was changed to α-MEM supplemented with 1%FBS. After 12 hours, trifluoroacetic acid or three peptides [(10 nmol/L PTH (1-34), 10 nmol/L G1R19 (1-34), and 100 nmol/L G1R19 (1-28)] were added into the culture medium. After 4 hours, the cells were washed gently ithcold PBS 3 times before total RNA was isolated. The expressions of Wnt related genes were measured by quantitative eal-time PCR. Results Most of the cells were polygonal and triangular; the cells were positive for ALP staining with blue cytoplasm at 14 days and the Al izarin red staining showed the formation of red mineral ized nodules in the special mineral ization induction medium at 28 days. The expressions of osteocalcin mRNA and Wnt5b mRNA in PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group were significantly higher than those in control group (P lt; 0.05); the expression of Wnt2 mRNA was significantly lower than that in control group (P lt; 0.05); the expression of β-catenin mRNA in PTH (1-34) group was significantly higher than that in control group (P lt; 0.05); the expression of Wnt7b mRNA in PTH (1-34) group and G1R19 (1- 34) group was higher than that in control group, and the G1R19 (1-34) group was higher than PTH (1-34) group and G1R19 (1-28) group (P lt; 0.05). Conclusion In the Wnt-related factors, PTH (1-34) and G1R19 (1-34) affect mainly canonical Wnt signal factors, but the G1R19 (1-28) chiefly acts on non-canonical Wnt signal factors.
Objective Metal wear products cause the aseptic loosening of joint prosthesis. To investigate the effect of metal ions Co2+ and Cr3+ on the osteoblast apoptosis, cell cycle distribution, and secretion of alkal ine phosphatase (ALP), and to search a method to prevent and treat aseptic loosening. Methods The mouse calvarial osteoblasts (MC3T3-E1) were cultured in vitro to 3-5 generations (5 × 105 cells/ mL) and divided into 2 groups: the experimental group and the controlgroup. The osteoblasts were cultured in α-MEM medium containing 10%FBS (the control group), and the mixed solution ofCoCl2 and CrCl3 was added after the osteoblasts cultured in α-MEM medium containing 10%FBS attached completely (the experimental group). At 12, 24, and 48 hours after culture, the osteoblast apoptosis and the cell cycle distribution were assessed by flow cytometry; and ELISA method was appl ied to detect ALP content in serum supernatant. Results At 12, 24, and 48 hours after culture, the apoptosis rates in the experimental group (13.90% ± 0.52%, 14.80% ± 0.41%, and 13.40% ± 0.26%) were significantly higher than those in the control group (8.56% ± 0.31%, 8.19% ± 0.24%, and 2.15% ± 0.11%), (P lt; 0.05); G2M (dividing phase) distribution ratio significantly decreased and G0G1 (dormancy stage) distribution ratio significantly increased when compared with those in the control group (P lt; 0.05); and the absorbency (A) values of ALP were 0.955 ± 0.052, 0.624 ± 0.041, and 0.498 ± 0.026 in the exprimental group, and were 1.664 ± 0.041, 1.986 ± 0.024, and 2.192 ± 0.041 in the control group, showing significant differences between 2 groups (P lt; 0.05). Conclusion Metal ions Co2+ and Cr3+ have a marked effect on osteoblasts cell cycle distribution, which can make most of the cells to be in dormancy stage (G0G1), up-regulate the apoptosis rate and inhibit the releasion of ALP from osteoblasts.
Objective As a bioactive material, the osteogenic activity of borate bioglass has been proved. To design a novel borate bioglass according to an improved formula and to investigate the effects of the borate bioglass on osteoblasts invitro for further research and potential cl inical appl ication. Methods The novel Na2O-K2O-MgO-CaO-P2O5-B2O3-SrO borate bioglass was prepared by melting process. The initial and secondary extracts were prepared according to ISO10993-12: 2007 respectively with different extract time of 0-24 hours and 24-48 hours. The osteoblasts (MC3T3-E1) of the 5th-15th passages from mouse were cocultured with the initial (initial extract group) and secondary (secondary extract group) extracts, respectively, to assess the effects of the borate bioglass on the cell prol iferation, protein synthesis, alkal ine phosphatase (ALP) activity, cell apoptosis, and cell migration; while α-MEM medium without addition of extract served as control group. Results The absorbance values at 450 nm were 0.356 0 ± 0.018 7, 0.331 0 ± 0.025 4, and 0.204 0 ± 0.013 8 in initial extract, secondary extract, and control groups, respectively, showing significant differences among 3 groups (P lt; 0.05). The total protein contents were (382.847 ± 9.521), (226.071 ± 5.847), and (220.248 ± 8.213) U in initial extract, secondary extract, and control groups, respectively; there were significant differences between initial extract group and control group, and between initial extract group and secondary group (P lt; 0.05), but there was no significant difference between secondary extract group and control group (P gt; 0.05). However, no significant difference was observed in the ALP activity [(0.013 01 ± 0.000 39), (0.012 93 ± 0.000 44), and (0.012 92 ± 0.000 35) U/ mg], apoptosis rate (7.03% ± 1.95%, 6.46% ± 2.88%, and 6.18% ± 2.21%), horizontal migration [(137.50 ± 11.43), (134.98 ± 10.50), (135.21 ± 8.66) μm], and transmembrane cell number [(10.92 ± 4.99), (10.07 ± 2.50), and (9.81 ± 2.64) cells/ field] among initial extract, secondary extract, and control groups (P gt; 0.05). Conclusion This novel borate bioglass has excellent cytocompatibil ity, which plays regulatory effects on the cell prol iferation, secretion, and migration.
Objective To review the regulation and mechanism of the microRNAs (miRNAs) in the bone and cartilage tissue. Methods Recent l iterature concerning the regulation and mechanism of the miRNAs in the bone and cartilage tissue was extensively reviewed, summarized, and analyzed. Results Recently miRNAs is a hot topic in the bone and cartilage tissue. More and more materials show its important regulatory role in osteogenesis and cartilage growth andregeneration, but the definite mechanisms have not been clear yet. Conclusion The study on miRNAs of bone and cartilage tissue can provide a new access to understanding the degenerative osteoarthritic diseases.
Objective To construct the eukaryotic expression vector of human bone morphogenetic protein 7 (hBMP-7) gene so as to observe its expression in rabbit adipose-derived stem cells (ADSCs) and its effects on osteogenic phe notype. Methods Several healthy 3-month-old Japanese rabbits of clean grade were chosen, female or male and weighing 3-4 kg. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by CD44, CD49d, andCD106 immunofluorescence staining. The eukaryotic expression vector of hBMP-7 gene (pcDNA3.1-hBMP-7) was constructed, which was transfected into rabbit ADSCs (3rd passage) by Li pofectamineTM 2000 after identified, then the expression of hBMP-7 in transfected ADSCs was detected. The alkal ine phosphatase (ALP) level and the collagen type I expression were detected by intracellular ALP spectrophotometry and immunofluorescence, respectively to assess the effect of hBMP-7 gene on the osteoblastic differentiation of ADSCs. Results ADSCs mostly presented fusiform and polygon shape with positive expressions of CD44 and CD49d and negative expression of CD106. The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene was successfully constructed and the expression of hBMP-7 was confirmed in ADSCs by immunohistochemical staining. The intracellular ALP quantitative detection showed that the activity of ALP was significantly higher in pcNDA3.1-hBMP-7 transfected group (experimental group) than in pcDNA3.1 transfected group (control group) at 7, 10, and 14 days after transfection (P lt; 0.05). The expression of collagen type I was higher in experimental group than in control group at 7 and 14 days after transfection (P lt; 0.05). Conclusion The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene is successfully constructed, which can express in ADSCs. The expressions of collagen type I and ALP in experimental group are higher than those in control group, which lays a basis for the local gene therapy of skeletal regeneration.
Objective Lots of metal ions accumulation and over-expression of receptor activator of NF-κB l igand (RANKL) around the prosthesis could be found in revision of total hip arthroplasty. To investigate the relationship between metal ions and aseptic loosening by observing the effects of Co2+ and Cr3+ ions on the expression of RANKL and osteoprotegerin(OPG) from osteoblast. Methods Osteoblasts were cultured in vitro at the density of 1 × 105 cells/mL, and were divided into 2 groups according to different culture solutions. In control group, osteoblasts were cultured with normal medium without CoCl2 and CrCl3. In experimental group, osteoblasts were cultured with the medium including CoCl2 (10 mg/ L) and CrCl3 (150 mg/L) solutions. The RT-PCR and ELISA methods were appl ied to detect the mRNA expression of RANKL and OPG and protein level at 24 and 48 hours after co-cultured, respectively. Results RT-PCR revealed that the mRNA expression of RANKL and OPG could be found in two groups at 24 and 48 hours after co-cultured, the expression was higher in the experimental group than in control group, especially the expression of RANKL, showing significant difference (P lt; 0.05). At 24 and 48 hours after co- cultured, the ratios of RANKL mRNA to OPG mRNA in the experimental group were 0.860 and 1.232, respectively, which were significantly higher than those in the control group (0.695 and 0.688,P lt; 0.05). ELISA revealed that the protein level of RANKL and OPG in experimental group were significantly higher than those in the control group (P lt; 0.05). Conclusion Co2+ and Cr3+ can stimulate the mRNA expressions of RANKL, OPG and secretion of those protein from osteoblasts, especially increase of the RANKL, which promotes the formation and activation of osteoblasts and the generation of aseptic loosening.
Objective To review the progress, controversy and trend in the regulation and mechanism of the microRNAs (miRNAs) during the osteogenesis. Methods Recent l iterature concerning regulation and mechanism of the miRNAs during the osteogenesis was extensively reviewed, summarized and analyzed. Results Recently miRNAs was a hot topic for osteogenesis. More and more materials showed its important role in ossification, but its definite mechanism was notclear. Conclusion Osteogenesis can be strengthened by miRNAs technology, which has a bright future and may also provide the molecular mechanism. The study on miRNAs of osteogenesis can provide a model to analyze and compare the osteogenetic effects of novel drugs.
Objective To study the effect of rat osteoblast conditioned culture medium on the BMSCs differentiation of allogeneic rat and to find a new approach to provide seed cells for bone tissue engineering. Methods BMSCs and osteoblasts were harvested from 10 healthy one-week-old SD rats (male and female, weighing 20-30 g) by adherent method and enzyme digestion method respectively. Cell identification was conducted. Osteoblast conditioned culture medium was prepared by mixing supernatant of osteoblasts at passage 1-5 with complete medium (1:1). Then, BMSCs at passage 2 were co-cultured with osteoblast conditioned culture medium (inducement group) and complete medium (control group), respectively. The morphological changes of co-cultured BMSCs were observed by inverted phase contrast microscope, the growth condition of BMSCs was detected by MTT method, the expressions of ALP, Col I and osteocalcin (OCN) in the cocultured BMSCs were tested by immunohistochemistry staining, and the expressions of Col I and OCN mRNA were detected by RT-PCR. Results In the inducement group, BMSCs grew bigger, changing from long fusiform to flat and polygon with protuberance 7 days after co-culture; the presence of cell colony-l ike growth was observed 9 days after co-culture. Cell growth curve demonstrated that the counts of BMSCs was increased with time, there were more cells in the control group than that of the inducement group, and there was a significant difference in cell counts between the control and the inducement group 4-7 days after co-culture (P lt; 0.05). For the inducement group, ALP staining was positive 12 days after co-culture, the calcium nodules were appeared 18 days after co-culture, Col I and OCN were positive 21 days after co-culture, and the expressions of Col I and OCN mRNA were detected by RT-PCR 21 days after co-culture. Conclusion Rat osteoblast conditioned culture medium can significantly induce the differentiation of allogeneic rats’ BMSCs towards osteoblasts.