The Preparation, Characterization And Bioactivity Evaluation Of Scaffold For Controlled Delivery Of Adrenomedullin In Vitro

Implant denture is regarded as"the third set of teeth"and has more irreplaceable advantages than traditional dental prosthesis. Not all the dentition defect cases suitable for implant restoration since some of them have the big problem of bone defect. There are many reasons that can cause bone defect, such as disuse atrophy of alveolar bone or external injury. Any of the factors can affect the initial stability of the implant by influencing the osteointegration. This problem can be solved by bone graft technical. But, to obtain the autogenous bone, we need to develop a second operation area, which patients can hardly accept since the extra trauma. At present, the researchers are focusing on gaining ideal synthetic bone scaffold materials, which provide support for the growth of new bone and occupy an important position in bone tissue engineering. Polymer scaffold materials have a lower biological activity and mechanical properties than inorganic scaffold materials and easy to cause aseptic inflammation. But inorganic scaffold materials also have a problem of how to promote bone cells to grow into them.Adrenomedullin (ADM) is a potent peptide with extensive biological effects that was originally isolated from pheochromocytoma by Japanese scholars. It is an effective mitogen of osteoblast, and also has a key role in vasculogenesis and takes part in many other functions in vivo. The role of ADM in bone metabolism is uncertain and some researchers considered that ADM has the mitosis promotion role on osteoblast and cartilage cell both in vitro and in vivo. Also, ADM has the role of inhibiting bone resorption in vivo, while its direct influence on osteoclast hasn't yet found. As a new found endothelial cell growth factor, ADM can promote vasculogenesis. ADM can stimulate the recovery of the hindlimb blood flow after ischemia-reperfusion in mice, which reflect the augmentation of secondary capillary density. So, ADM plays a significant role by promoting vasculogenesis in the reconstruction of soft and hard tissue.Whether the synthetic bone scaffold materials have both promoting bone formation effect and vasculogenesis activity after combine with ADM is uncertain from the report of domestic and international researchers.In our research we evaluated the effect of PLGA/nano-hydroxyapatite scaffold containing chitosan microspheres for controlled delivery of ADM on the proliferation and differentiation of MG-63 and HUVEC cells in vitro. We used real time PCR to detect the characteristic of the synthetic scaffold and the influence of ADM on bone formation and vasculogenesis related genes. And then, their influence on Collagen I, Runx2 and VEGF protein was detected by Western Blot. Our results provide theoretical basis for clinical application in the future.1. The influence of different density ADM on the proliferation and differentiation of MG63 and HUVECObjective: Osteoblast-like cells MG63 was used as the model of human osteoblast and HUVEC was used as the model of vascular endothelial cell to evaluate the influence of ADM on their biological characteristic.Methods: MG63 and HUVEC cells were cultured in high goucose DMEM and IMDM respectively in 96 well plates and both medium contain 10% fetal bovine serum. Put 1μL gradient concentration ADM in and make the final concentration to 10^-13mol·L^-1-10^-7mol·L^-1. Put equal amount of PBS in the control group and check them in the 1^st day, 2nd day, 3^rd day, 4th day and 5^th day. To find the optimal density of ADM that is conducive to the proliferation and differentiation of MG63 and HUVEC, we detected the proliferation activity of MG63 and HUVEC cells by MTT and the differentiation activity of MG63 cells by ALP activity test.Results: ADM has the strongest ability to promote MG63 proliferation when the density is 10^-9M and 10^-8M while in HUVEC the density is 10^-8M. In the test of ALP activity, the strongest promotion ability on the differentiation activity of MG63 cells was appeared when the density of ADM was 10^-8M and 10^-7M. Thus, we will choose 10^-8 M as the optimal ADM density in our following study.2. The preparation, characterization and degradation of PLGA/nano-hydroxyapatite scaffold containing chitosan microspheres for controlled delivery of mutifucational peptide–adrenomedullinObjective: To investigate the possibility of adrenomedullin loaded into chitosan microspheres and prepare the scaffold that containing microspheres. Study the characterization, mechanical function and degradation ability of the microspheres and the scaffold in vitro and in vivo.Methods: In the presence of tripolyphosphate (TPP), chitosan microspheres (CMs) loaded with adrenomedullin (ADM) were prepared by an emulsion-ionic cross-linking method. Then, PLGA/ nano-hydroxyapatite scaffold that containing microspheres was developed by thermally induced phase separation. Scanning electron microscope was used to analyze the characterization of the microspheres and scaffold. The material degradation have been tested within 12 weeks in vitro and in vivo. To study whether there has any changes in the nature performance of the material after loaded with microspheres, we detected the mechanical function by compress experiment.Results: The diameter of the microspheres was well-distributed and has the Gaussian distribution with the average diameter of 42.69μm. PLGA/nHA scaffold material has the typical polymerizer poriform structure with the pore size of 50-220μm and the pores were interconnected. The drug-loading rate was 0.058% and the encapsulation efficiency was 79.4% after calculated by HPLC determination. The initial burst release of microspheres loading with adrenomedullin in the first three days was 50.7% and slowed down in the forth day, then reached 68.3% in the 10th day. The release of ADM from PLGA/nHA/CMs scaffold material was 21.0% in the first week and shown a linearity slow release in 2-4 weeks, then reach the total release amount of 51.8% in the 12th week. The interval porosity of the blank scaffold material was 90.8% and the interval porosity after loaded 30% chitosan microspheres can also reach 88.9%. The density of the scaffold with no microsphere was (0.045±0.017)g/mL, which was significantly lower than the density of scaffold containing 30% chitosan microspheres (0.083±0.020)g/mL (P<0.05). The degradation of PLGA/nHA/CMs scaffold material was slightly faster in the first three weeks and then reaches a linearity mode by in vitro weight loss testing. The degradation of PLGA/nHA/CMs scaffold material was 12.23% at the end of 12 weeks while the weight loss of PLGA/nHA scaffold material was 8.27%. The PH of incubation buffer for PLGA/nHA/CMs scaffold material was 6.88±0.12 and for PLGA/nHA scaffold material was 6.49±0.09 at the end of 12 weeks. As for the absorption rate, the result shown that the rate of PLGA/nHA/CMs scaffold material was 66.9% in the first week and gradually rises till stable in 82.15% in the 6th week and reach 88.34% in the 12th week. The weight loss rate of PLGA/nHA/CMs scaffold material after degradation in vivo was faster in the first four weeks and reach 10.7% at the end of the forth week. The rate was tended to be mild in 4-8 weeks and shown a rise period in 8-12 weeks, and then reached 21.4% in the 12th week. The compress experiment shown the compressive strength of the scaffold loaded 30% chitosan microspheres have been improved significantly. The compressive strength of PLGA/nHA/CMs scaffold material was (1.54±0.20)MPa and the compressive modulus was rised to (7.24±0.42)MPa. 3. The influence of drug-loading scaffold material on the proliferation and differentiation of MG-63 and HUVEC cellsObjective: Osteoblast-like cells MG63 was used as the model of osteoblast and use HUVEC as the model of vascular endothelial cell to evaluate the influence of drug-loading scaffold material and blank scaffold material on their biological characteristic.Methods: MG63 and HUVEC cells were conventional cultured and put into cell culture plate. PLGA/nHA/CMs/ADM was the experimental group, PLGA/nHA/CMs were the control group and the cells were the blank group. Evaluate the hemolysis characteristic and the biological activity of the drug-loading scaffold material by hemolysis test, ALP activity test, fluorescein stain of scaffold surface cell and MTT.Results: The hemolysis rate of the PLGA/nHA/CMs was 0.171% and far less than 5%, which close to the blank group. The results shown that the PLGA/nHA/CMs scaffold was safe to be used and had no acute hemolysis activity. The drug-loading scaffold and scaffold itself could accelerate the proliferation of osteoblast-like cells and vascular endothelial cells and the differentiation of osteoblast-like cells4. The influence of ADM-loading scaffold material on the related functional gene and protein of MG63 and HUVEC cellsObjective: an Osteoblast-like cell MG63 was used as the model of osteoblast and use HUVEC as the model of vascular endothelial cell to evaluate the influence of drug-loading scaffold material on their bone formation and vascularize related gene and protein expression.Methods: MG63 and HUVEC cells were conventional cultured and put onto the surface of the scaffold material. PLGA/nHA/CMs/ADM was the experimental group, PLGA/nHA/CMs were the control group and the cells were the blank group. Extract RNA and protein after cellula digestivum in the 1^st day, 3^rd day and 5^th day. Detect the expression of COL1α1, Runx2, SP7, OPN, VEGF and RAMP2 gene and the expression of VEGF, Collagen I and RUNX2 protein.Results: The results of Real-time PCR showed that the expression of Runx2 mRNA in MG63 cells in experimental group was slightly higher than the control group and significantly higher than the blank group in the 1^st day. The expression of Runx2 mRNA was significantly up-regulated in the experimental group and the control group in the 3^rd day. The mRNA expression of the three groups was all down-regulated in the 5^th day and was slightly higher in the experimental group than in the blank group. The expression of SP7 mRNA in the experimental group was a little lower than the control group and at the same level of the blank group in the 1^st day. While in the 3^rd day, the SP7 mRNA expression was significantly up-regulated in the experimental group and higher than the other two groups. The SP7 mRNA expression was even higher in the experimental group in the 5^th day, which was 2.7 times higher than the control group and 4.68 times higher than the blank group. The control group was 1.73 times higher than the blank group. The expression of OPN mRNA in the control group was obviously increased in the 1^st day and the same result was got in the 3^rd day, while in the 5^th day there's no evident difference in the three groups. The COL1α1 mRNA expression in the experimental group was obviously higher than the other two groups in the 3^rd and 5^th day and the expression was in the same level in the control group and the blank group.The expression of RAMP2 gene in HUVEC cells in the experimental group was slightly lower than the control group but higher than the blank group in the 1^st day. The expression was obviously increased in the experimental group, which was 2.3 times higher than the control group and 2.4 times higher than the blank group in the 3^rd day. In the 5^th day, the gene expression in experimental group was 2.06 times higher than the control group and 2.57 times higher than the blank group. The expression of VEGF gene in the experimental group was significantly higher than the other two groups in the 1^st day, 3^rd day and 5^th day. It's 1.78 times than the control group and 1.68 times higher than the blank group in the 1^st day; 1.55 times than the control group and 2.1 times higher than the blank group in the 3^rd day; 1.88 times than the control group and 2.32 times higher than the blank group in the 5^th day. There's no obvious difference between the control group and the blank group in the 1^st day and the VEGF expression in the control group was slightly lower than the blank group.The expression of Runx2 protein was obviously higher in experimental group than the other two groups in the 1^st day after detected by Western Blot. The protein expression was significantly higher in the experimental group than the blank group, while was significantly lower in the control group than the blank group in the 3^rd day. In the 5^th day, the protein expression has no significantly difference between the experimental group and the control group, while the blank group was significantly lower than the other two groups.Detected by Western Blot, the Collagen I protein expression in the experimental group and the control group was significantly higher than the blank group in the 1^st day. There's no obvious difference in the experimental group and the blank group in the 3^rd day, which was the same with their mRNA level. The expression of VEGF protein in the experimental group and the control group was significantly higher than the blank group in the 1^st day after detected by Western Blot. There's no significant difference among the three groups in the 3^rd day and the protein expression in the experimental group was significantly higher than the other two groups.In conclusion, ADM has a noticeable promoting effect on the proliferation and differentiation of osteoblast and vascular endothelial cell. PLGA/nano-hydroxyapatite scaffold containing chitosan microspheres for controlled delivery of adrenomedullin also has the promoting effect on these two kinds of cells and can promote the bone formation and vascularization related gene and protein expression in the early stage. This research provide theoretical basis for the dental implant clinical application of controlled delivery of adrenomedullin scaffold.