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 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 To investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.
Objective To investigate the effect of 1,25(OH)2VD3 on differentiation of embryonic stem cells (ESCs) into osteoblasts. Methods Osteoblasts were isolated and cultured from calvarium of 2-day-old Kunming white mice, embryoid bodies (EBs) were prepared with modified zur Nieden method. EBs were divided into 4 groups according to different mediums: group A, as the control group, in which EBs medium contained no leukemia inhibitory factor; group B, in which EBs medium contained supplements of Vitamin C (VC, 50 μg/mL) and β-glycerophosphate (β-GP, 50 mmol/L); group C, inwhich EBs medium was the same as that of group B and 5 × 104 osteoblasts of 3rd passage were seeded into each well; group D, in which the medium contained supplements of VC (50 μg/mL), β-GP (50 mmol/L) and 1,25(OH)2VD(4 × 10-9 mol/L), and 5 × 104 osteoblasts of 3rd passage were seeded into each well. The ALP activity was determined by ALP reagent kit every 5 days. The RQ-PCR was performed to measure the mRNA expressions of osteocalcin (OCN). Al izarin red S staining was performed to count the bone nodules. Results The expression of ALP witnessed no obvious change in each group within 5 days after adherence of EBs, but increased gradually after 5 days. The expression of ALP in group D reached the peak at 20 days. Red nodules with clear outl ine and different sizes were evident by microscope. Al izarin red S staining testified the number of bone noudles in groups A, B, C and D was 20 ± 8, 18 ± 5, 31 ± 1 and 50 ± 1, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). The result of RQ-PCR showed that the mRNA expressions of OCN in groups A, B, C and D was 10.18 ± 1.17, 20.29 ± 1.03, 18.84 ± 4.07 and 32.15 ± 5.23, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). Conclusion The combined action of 1,25(OH)2VD(4 × 10-9 mol/L), VC, and β-GP can effectively promote the differentiation of the ESCs-derived osteoblasts.
Objective To review the progress, methods and obstacles in the differentiation of embryonic stem cells(ESCs) into osteoblasts in vitro. Methods The recent literature concerned with the differentiation of ESCs into the osteoblasts was extensively reviewed and briefly summarized. Results ESCs was a good tool for derivation of obsteoblasts.Conclusion The study on the induction of ESCsinto the osteogenic lineage provides a model for analyzing the molecular processes of osteoblasts development in vivo and establishes the foundation for the use of ESCs in skeletal tissue repair.
Objective To study the function of platelet-derived growth factor (PDGF) in inducing phosphorylation extracellular signalregulated kinase 1/2 (pERK1/2) localization in osteoblasts. Methods Primary osteoblasts were isolated and cultured from cranial bone of 10 mice atthe age of 3 days, weighting 6-9 g without limitation in male and female.The sixth passage osteoblasts were incubated in 1% serum for 12 hours and divided into 2 groups: treated with DMSO(control group) or with PP2(experimentalgroup) for 30 minutes. Each group was further divided into 2 subgroups according to with or without PDGF (20ng/ml) stimulation for 10 minutes. pERK1/2 localization was analysized by immunofluorescence staining in osteoblasts pretreated with or without Src inhibitor PP2. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into two subgroupsaccording to with or without PDGF (20 ng/ml) stimulation for 10 mintues. The ability of osteoblast migration was determined by wound healing assay. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or 10 μmol/L PP2 (experimental group) for 30 mintues. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation.The pERK1/2 was determined by Western blot in osteoblastic cytoskeleton inducedby PDGF. Results Immunofluorescence staining showed pERK1/2 localization in osteoblastic nuclears and focal adhesions after PDGF stimulation. PP2 significantly inhibited ERK1/2 localization in focal adhesions, but not in nuclears. The wound healing assay results showed that PP2 significantly inhibited osteoblast migration induced by PDGF. The result of Western blot demonstrated that pERK1/2 in osteoblastic cytoskeleton was significantly inhibitedSrc activation is required for pERK1/2 translocalization to focal adhesions and osteoblasts migration.
Objective To review the research progress of osteoblasts in the hematopoietic microenvironment of bone marrow and regulatory pathways and mechanisms. Methods The advances in the osteoblasts as crucial components for hematopoietic microenvironment in bone marrow, regulation to osteoblasts and hematopoietic stem cells(HSCs), and correlative singal pathways and mechanisms were introduced based on the recent related literature. Results Evidence indicates that osteoblasts are crucial components of the hematopoietic microenvironments in adult bone marrow. The osteoblasts maintainthe quiescence of primitive HSCs by the signaling receptorsligands, secreted cell factors and celladhesion molecules and by regulating other cells in the niche. The quiescent primitive HSCs persist stem cell characteristic which has unlimited selfrenewal and multipotent differentiation potential. Conclusion The further understanding of the relationship between osteoblasts and hematopoietic microenvironment should lead to development of new strategies directed toward clinical therapeutics of HSCs transplantation.
Objective To find a new culture system to induce proliferation and osteodifferentiation of marrow stromal cells (MSCs) in vitro for bone tissueeng ineering． Methods There were four groups in this experiment to study effects of Passage 3 osteoblasts derived from the rat cranium and the osteogenic inductor (1 nmol/L dexamethasone，10 mmol/L beta-glycero-phosphate，50 μg/ml retin oic acid) on growth of MSCs isolated from the rat femur and the tibia． MSCs were cultured in the DMEM medium (the c ontrol group) and in the osteoinductive culture medium (the inductor group)；fur thermore, MSCs were co-cultured with the osteoblasts in the DMEM medium (the osteoblast group) and in the osteoinductive culture medium (the combined treatment group)．The cells in the four groups were counted every 2 days for 8 days and alkaline phosphatase (ALP) activity of MSCs at 10 days of cultivation was measured．The MRNA expression of osteocalcin (OC) of MSCs at 2 weeks was assayed with the reverse transcript polymase chain reaction (RT-PCR)． Results There were more cells in the osteoblast group than in the control group(31.73±3.31×104 V S. 24.33±3.04×104, Plt;0.05), but there were fewer cells in the inductor gro up(16.23±2.44×104, Plt;0.05). There was no significant difference in th e cell number between the combined treatment group (21.54±2.29×104) and th e control group（Pgt;0.05）．The ALP activity was higher in the combined trea tment group （2.01±0.56 U）than in the control group (1.27±0.43 U), in the inductor group（1.27±0.43 U）, and in the osteoblast group （0.77±0.19 U）.The osteocalcin mRNA was expressed in the three treat ment groups but was not expressed in the control group. The significantly higher leve l of the osteocalcin mRNA was expressed in the inductor group（0.783±0.094）and in the combined treatment group（0.814±0.071）than in the osteoblast group（0.302±0.026） (Plt;0.05). Conclusion The combined use of t he osteoblast and the inductor can induce marrow stromal cells. Their combined u se does not affect the normal proliferation but can obviously promote the osteodifferentiation of marrow stromal cells. This combined use can become a new culture system of the seed cells for bone tissue engineering．
Objective To observe effects of the core binding factor α1 (Cbfα1) in its promoting differentiation of the rabbit marrow mesenchym al stem cells (MSCs) into osteoblasts. Methods The rabbit marrow MSCs were isolated and cult ured in vitro and were divided into 3 groups. In the control group, the marr ow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and βGP 10 mmol/L); and in the experimental group , the ywere transfected with AdEasy1/Cbfα1,and then were cultured by the condition m edium. The alkaline phosphatase(ALP) activity and the experission of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohi stochemical methods at 3 days，1，2，3，and 4 weeks after inducement. Results More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfectived with AdEasy1/Cbfα1. The ALP activity and the experission of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks （Plt;0.05）.The mineralized node began to appear at 2 weeks in the experiment al group and the single induction group, but did not appear in control group. Conclusion Cbfα1 can obviously promote differentiation of the rabb it marrow mesenchymal stem cells into the osteoblasts.
Objective To investigate the effect of WO-1 on the proliferation and differentiation of human embryonic osteoblasts (HEO) and to provide research methods of bone tissue engineering. Methods HEO were isolated from periosteum and calvaria and then cultrued in vitro. The doseeffect relationship between WO-1 concentration and biological effect of HEO was evaluated by growth curve and 3 H-TdR count. The effect of WO-1 on cell activity and proliferation was investigated by cloning efficiency,cell cycle analysis was determined by flow cytometer and morphological was examined through transmission electron microscope. Moreover, the effect of WO-1 on osteoblastic function was evaluated at protein and mRNA levels by ALP activity, 3 H-proline incorporation, osteocalcin secretion (RIA) and mRNA expression of type I collagen and osteocalcin (RT-PCR). Results The proliferation of HEO was inhibited in high concentration of WO-1,while it was promoted in low concentration of WO-1. The optimal dose was 8 μg/ml, and there was dose-effect relationship in the certain range of WO-1 concentration (0.25 μg/ml to 8 μg/ml). In 8 μg/ml of WO-1, the cloning efficiency and cloning volume of HEO were inereased, population doubling time was decreased.All indexes of ostoblastic function including ALP activity, type I collagen synthesis and osteocalcin secertion were inereased, the more sufficed cell organs were observed under transmission electron microscope than control group(P＜0.05). Conclusion WO-1 can promote the cell activity and proliferation of HEO cultured in vitro inlow concentration, enhance the synthesis of extracellular mamix, such as type Icollagen and osteocalcin, and accelerate the mineralization of osteoid. WO-1 can be used as a stimulant of proliferation and differentiation of HEO in the research of bone tissue engineering, which provide the theoretical basis in clinical application.