| BACKGROUNDNerves and blood vessels take part in bone formation, development and function simultaneously. Engineered bone can become mature bone tissue if it has the function of secrete, metabolism and promoting hematopoiesis normally. At present there are few researches on the relationship between nerves and bone often concerning single factor. But the constructure of engineered bone in vivo is due to the effect of a variety of seed cells. Nerve affected blood vessels to affect osteogenesis indirectly. Interaction between nerves and blood vessels is quite complicated and results of the current researches are also inconsistent at present. In this study, the relationship between seed cells for nerves and vessels has been researched to find some factors from host on promoting angiogenesis, to optimize osteogenic effect locally and provide experimental basis for neuralized and vascularized bone tissue engineering in future.OBJECT1. To study the effects of SCs to vascular endothelial cells of different sources on proliferation, migration, secretion and formation of blood vessels.2. To study the promoting mechanism of SCs to BMSCs derived vascular endothelial cells for formation of blood vessels.3. To study the local bone formed effects under the action of SCs and vascular endothelial cells.MATERIAL AND METHODS1. Isolation, culture and identification of rat BMSCs, AECs and SCs and endothelial differentiation of BMSCsRat BMSCs were isolated and cultured by whole marrow method and identified by flow surface antigen detection and osteogenic, chondrogenic and adipogenic differentiation. Rat AECs were isolated by velles intimal digestion method and cultured and purified by M199 medium with VEGF and heparin. AECs were identified by Vwf and Pecam-1 IF. Rat SCs were isolated by tissue adherence method and cultured and purified by low glucose DMEM medium with Forskolin and HRG1-β. SCs were identified by S-100 IHC. BMSCs were induced to vascular endothelial cells (IECs) by high glucose DMEM medium with VEGF, EGF, FGF-B and R3-IGF-1 (EGM-2). IECs were identified by Vwf and Pecam-1 IF, TEM, Vwf, CD34, Kdr, Nos2 and Nos3 mRNA expression compared with AECs and BMSCs. The mRNA relative expression data of each target gene was analyzed by SPSS 16.0 statistical software, P value< 0.05 was regarded significant difference. The comparison of each group was analysized with One-way ANOVA, multiple comparisons between groups using LSD method.2. The primary mechanism of SCs promoting proliferation, migration, secretion and the function of vessels formation in vivo and vitroSCs conditioned medium (SC-CM) were collected and used to stimulate and culture IECs or AECs as the experimental group. Regular culture medium without growth factors were used to culture IECs or AECs as the control group. Cells count was detected by CyQuant assay kit on 1 d,3d,5 d,7 d,9 d and 11 d. VEGF concentration in SC-CM was detected by Elisa. For transwell coculture system, SCs were seeded in lower layer as the experimental group, and no cells seeded as the control group. IECs or AECs were seeded in upper layer and cells migrated were observed after 16 h. The cells migrated were stained with crystal violet and extracted with acid elution to determine OD of extracted fluid of each group. Pecam-1 and CD34 mRNA relative expression of IECs or AECs cocultured with SCs were detected by qRT-PCR at 1 d, 3d,7 d and 10 d. The medium in coculture system were collected and detected for NO concentration on 1 d,3d,5 d and 7 d. Nos2 and Nos3 mRNA relative expression of IECs or AECs cocultured with SCs were detected by qRT-PCR at 1 d,3d,7 d and 10 d. SC-CM were used to stimulate and culture IECs or AECs as the experimental group. Regular culture medium without growth factors were used to culture IECs or AECs as the control group. The vessels-like formation in matrigen was observed after 6 h. Using gelatin as a carrier, SCs and IECs in SC-CM-DMEM were injected into rat corneal basal layer as the experimental group, and only IECs-gelatin as the control group. Ink was infused from aorta and angiogenesis at the injection site in corneal and the results were observed after 12 d. The comparison of cells count and OD of each group was analyzed with two independent samples t test. The mRNA relative expression data of each target gene was analyzed by SPSS 16.0 statistical software, P value<0.05 was regarded significant difference. The comparison of each group was analyzed with One-way ANOVA, multiple comparisons between groups using LSD method.3. Mechanism of SCs promoting vessel formation and BMSCs osteogenic differentiationSC-CM were used to stimulate and culture IECs or AECs as the experimental group. Regular culture medium without growth factors were used to culture IECs or AECs as the control group. VEGF concentration in medium of each group was detected by Elisa at 1 d,3 d, 7 d and 10 d. Nestin expression of IECs and AECs were detected by IF. IECs coculturing with SCs as the experimental group and IECs without coculture as the control group, nestin mRNA relative expression of IECs or AECs cocultured with SCs were detected by qRT-PCR at 1 d,3d,7 d and 10 d. SC-CM were used to stimulate and be used to culture IECs or AECs as the experimental group. Regular culture medium without growth factors were used to culture IECs or AECs as the control group. VEGFR1 and nestin expression simultaneously of IECs and AECs were detected by double-marked IF. IECs coculturing with SCs as the experimental group and SCs without coculture as the control group. Mmpl4 and Timp2 mRNA relative expression of SCs were detected by qRT-PCR at 1 d,3d,7 d and 10 d. Nestin, VEGFR1 and VEGFR2 expression simultaneously were detected with Western blot. The SC-CM medium culturing IECs or AECs were collected at 1 d,3d,7 d and 10 d, and Timp2 expression in medium were detected with Western blot. BMSCs coculturing with IECs in SC-CM-osteogenic induced medium as the experimental group 1, and BMSCs coculturing with IECs in osteogenic induced medium as the experimental group 2, and BMSCs culturing in osteogenic induced medium as the control group, ALP, Bglap, Bmp2, Colla and Spp mRNA relative expression of BMSCs were detected by qRT-PCR at 4 d,7 d,14 d-and 21 d. The comparison of each group was analyzed with One-way ANOVA, multiple comparisons between groups using LSD method. P value< 0.05 was regarded significant difference.4. The early construction of tissue engineered bone in vivo with pre-seeding IECs and SCs on p-TCP scaffoldViral vector expressing eGFP and RFP was transfected BMSCs and marked BMSCs were induced to osteoblasts (IOB) and IECs respectively. SCs were marked with Hoechst 33342. IECs, AECs or SCs were seeded on p-TCP scaffold and the adherence situation was observed with fluorescence microscope and SEM. SCs and IECs were pre-seeded on P-TCP scaffold by the negative pressure method, and IOB were seeded 3 days later as the experimental group 1. IECs were pre-seeded on β-TCP scaffold by the negative pressure method, and IOB were seeded 3 days later as the experimental group 2. Only IOB were seeded as the control group. The scaffolds with cells were implanted into rat 6 mm long femur segmental bone defect. X ray, HE or Masson staining and MicroCT scanning were used to detect the osteogenic effects locally.RESULTS1. BMSCs proliferated actively after 3 rd passage, showing spindle-like and limpid cell membrane. BMSCs grew parallelly or swirly and were capable to induced to osetoblasts, chondrocyte and lipoblast. Flow cytometry showed BMSCs could express CD29 and CD44, not CD34 or CD45. AECs were polygonal or oval and cell boundary was unclear, showing cobblestone-like appearance while fusion. Vwf and Pecam-1 IF of AECs were positive. SCs had quite narrow cell bodies and were bipolar or unipolar. S-100 IHC of SCs was positive. IECs became blunt and also showed cobblestone-like appearance. Vwf and Pecam-1 IF of IECs were positive. TEM showed IECs became blunt and nucleus became bigger. W-P bodies appeared near nucleus. qRT-PCR of IECs showed Vwf, CD34, Kdr, Nos2 and Nos3 mRNA expression compared with AECs and BMSCs increased significantly.2. IECs count was higher significantly of the experimental group than the control group after the 3 rd day. AECs count of the experimental group was higher significantly than the control group only at the 3 rd day, and lower at other times. VEGF concentration of SC-CM was 499.850 pg/mL and 865.520 pg/mL. VEGF levels in medium of the control group could be regarded as 0. IECs migrated of the experimental group were more than the control group. OD of the extracted fluid of the experimental group was higher significantly than the control group. AECs had no significant difference between the experimental and control group. Pecam-1 mRNA expression of IECs was higher significantly of the experimental group than the control group at the 7 th day. CD34 mRNA expression of IECs was higher significantly of the experimental group than the control group at each time (P< 0.05) and showed increased trend with the time. NO released of IECs of the experimental group was higher significantly than the control group at each time (P< 0.05) and showed increased trend with the time after 3 d. NO released of AECs was significantly lower of the experimental group than the control group at each time(P <0.05) and showed decreased trend of each group. Nos2 mRNA expression of IECs was higher significantly of the experimental group than the control group (P<0.05). Nos3 mRNA expression of IECs of the experimental group was higher significantly than the control group except at 10 d (P<0.05). More tube-like structures appeared in vitro of the IECs experimental group than the control group. But there was no difference between the AECs experimental group and control group. More vessels could be observed in vivo of the experimental group than the control group at the 12 th day.3. VEGF concentration of the experimental group was higher than the control group for IECs and showed the increased trend. It was also higher for AECs, but the increased trend was not apparente and the total concentration was lower than IECs. Nestin IF was positive to IECs,but negative to AECs. Nestin mRNA expression of IECs of the experimental group was higher significantly than the control group at each time and showed an increased trend (P<0.05). Double-marked IF showed nestin and VEGFR1 of the experimental group could express strongly simutaneously and both of them were stronger than the control group. Mmpl4 mRNA expression of SCs of the experimental IECs group was higher significantly than the control IECs group, and lower for Timp2 at each time and the results on Timp2 showed an decreased trend (P<0.05). Mmpl4 mRNA expression of SCs of the experimental AECs group was lower significantly than the control AECs group, and lower for Timp2 at each time except at the 7 th day (P<0.05). Western blot showed that nestin, VEGFR1 and VEGFR2 expression of the IECs experimental group had an increased trend. All target protein expression were not apparent before the 3 rd day and became stronger than the control group. The experimental AECs group had no nestin expression and VEGFRl expression were weaker than the control AECs group. VEGFR2 expression were similar for each group and each time. Timp2 expression of SC-CM-DMEM was weaker than SC-CM-M199. ALP, Bglap, Bmp2, Colla and Spp mRNA expression of BMSCs showed significant difference among the experimental 1 group, the experimental 2 group and the control group, and the sequence from low to high was the experimental 1 group, the experimental 2 group and the control group (P<0.05).4. eGFP-BMSCs or RFP-BMSCs was capable of being induced to osteoblasts or vascular endothelial cells respectively. And ALP staning, Vwf and Pecam-1 IF showed positive. The morphology and viability of SCs were good marked by Hoechst 33342. Fluorescence microscopy and SEM showed 3 kinds of cells could adhere well onto the scaffold. The process of surgical implantation of fixation plate and the scaffold with cells was under good condition. HE staining at 6 w showed that some bone tissue grew into the scaffold and osteoblasts and hypertrophic cartilage cells distributed generally at the area where the scaffold had been absorbed in the experimental 1 group; the constructure of the scaffold still appeared and no bone tissue could be found and much fibrous tissue grew into the porous of the scaffold with few osteoblasts and cartilage cells in the experimental 2 group and the control group. Masson staining at 12 w showed that the scaffold had been absorbed generally with amount of osteoblasts and cartilage cells and the collagen had been fused with the implant widely in the experimental 1 group;the scaffold had generated locally with some newborn collagen from bone formation in the experimental 2 group and osteoblasts and chondrocytes were fewer than the experimental 2 group; some fibrous tissue grew into the scaffold without newborn bone collagen. MicroCT scanning at 12 w showed that much newborn bone and bone callus appeared at the bone defect and bridged between the ends of fracture bone with healing of bone defect in the experimental 1 group; some bone callus appeared on the superficial parts of the bone defect with only connecting with the ends of the fracture bone in the experimental 2 group; few callus could be observed without healing of the bone defect in the control group.CONCLUSION1. SCs can promote the proliferation of IECs and have the same effects on AECs at the early stage, but the effects can change into inhibition at the late stage. SCs can promote the migration and NO release of IECs, due to the capcability of SCs on secreting VEGF, upregulating Pecam-1 and CD34 expression and promoting the activity of nitric oxide synthase (NOS).2. SCs can upregulate nestin expression of IECs to promote VEGFR expression. IECs can promote Mmp14 expression and inhibit Timp2 expression to enhance the endothelial differentiation of BMSCs. SCs and IECs can delay the process of osteogenic differentiation of BMSCs at the early stage.3. Pre-seeding IECs into scaffolds can improve the bone formation efficiency in vivo. The effect of promoting angiogenesis of IECs due to SCs may play an apparent role at the early stage of osteogenesis in vivo. |
