Preparation Of Porous N-HA/PGS -M Composite Scaffold And Experimental Research On Its Material And Biological Characteristics In Vitro And In Vivo

Background:The treatment of bone defects has been a major clinical problem for orthopedic surgeons.Although autogenous bone graftand allogenic bone graft have good clinical results for patients,their limitation in donor bone stocks and complications at the site of extraction limit their clinical application.The development of bone tissue engineering and other disciplines in the improvement and development of artificial synthesis or natural materials provides hope for bone defect.Composite scaffolds made from synthetic organic and natural inorganic materials have many performance advantages,such as good biocompatibility,high degradability and toughness,which are widely valued by researchers and clinical workers.Synthetic polymer materials have good mechanical properties,and degradable polymer materials degrade directly in the body after completing their functions.Poly glycerol sebacate?PGS?is a degradable polymer material with good compatibility and bioactivity which is widely used in clinical research.Maleic anhydride?MAH?can promote the adhesion of osteoblasts by providing carboxyl and hydroxyl groups to promote tissue formation and growth,and improve the performance of the compound.Nano hydroxyapatite?n-HA?,as the most important inorganic component of bone and teeth,will not cause obvious rejection reaction,but its application is limited due to its brittleness.A new biodegradable polymer material?PGS-M?was prepared by grafting PGS with Maleic anhydride,which promoted osteoblast adhesion,proliferation and differentiation,and further prepared PGS-M-n-HA composite scaffolds.The advantages of the composite scaffold material make up for the lack of mechanical properties of PGS-M and improve biological performance of n-HA.Methods:PGS,PGS-M and PGS-M-n-HA were prepared by high temperature,vacuum and other steps.The chemical structures of PGS-M and PGS-M-n-HA were analyzed by H-NMR and Fourier transform infrared spectroscopy.Morphological characterization and porosity of PGS-M and PGS-M-n-HA were observed with scanning electron microscope.Differential scanning calorimeter and thermal analysis system were used to test the thermal performance of PGS-M and PGS-M-n-HA.The degradability of PGS-M and PGS-M-n-HA were tested with weight loss method after immerse.The mechanical properties of PGS-M and PGS-M-n-HA were tested with a universal material testing machine.CCK8 was used to detect the effects of PGS-M and PGS-M-n-HA on the proliferation ability of AD-MSCs.The effects of PGS-M and PGS-M-n-HA on the expression of osteogenic differentiation associated genes of AD-MSCs were detected by QPCR,WB and IF.The effects of PGS-M and PGS-M-n-HA on the expression of inflammatory factors in AD-MSCs were detected by QPCR.Scanning electron microscopy and energy dispersive X-ray spectroscopy were used to detect the calcium and phosphorus precipitation ability of PGS-M and PGS-M-n-HA.The osteogenic induction and differentiation ability of PGS-M and PGS-M-n-HA in vivo was detected by three-dimensional CT and histology of rat skull defect model..Results:After the preparation of PGS-M,the proton signals of the methylene?CH2?and glycerol portions of PGS and the proton signals of the double bond on maleic acid were detected.43%of the hydroxyl groups were replaced by maleic anhydride.After the preparation of PGS-M-n-HA-0.4,PGS-M-n-HA-0.5,and PGS-M-n-HA-0.6,hydroxyl groups formed by n-HA C-O were detected at an absorption peak at 1036 cm^-1.According to the SEM picture,the pore diameter of porous scaffolds can reach 150-300?m.The porosity of the porous scaffold is extremely large,and a large number of holes inside of the scaffold are also conducive to cell migration and adhesion.The porosity of the PGS-M-n-HA-0.4,PGS-M-n-HA-0.5,and PGS-M-n-HA-0.6series scaffolds were 90.13%,88.21%,and 84.56%,respectively.The Glass temperature?Tg?of the PGS-M and PGS-M-n-HA scaffold is between-25^oC and-30^oC,which indicates that the scaffold is in a high elastic state at room temperature and body temperature,and can exhibit good elasticity.In the test temperature range?-50^oC–150^oC?,no crystallization peaks,oxidation peaksand melting peaks of the scaffold were observed.The weight of the PGS-M and PGS-M-n-HA series scaffolds begin to decrease at about heating to250^oC,and the weight of PGS-M decrease 100%and PGS-M-n-HA series scaffolds decrease 40%to 60%at the temperature of 600 ^oC.The degradation of PGS-M and PGS-M-n-HA series scaffolds showed a linear relationship.At 8week,PGS-M?PGS-M-n-HA-0.4?PGS-M-n-HA-0.5?PGS-M-n-HA-0.6scaffolds degraded by 33.26%,25.21%,21.57%and 16.34%respectively.The compressive strength of PGS-M-n-HA-0.4,PGS-M-n-HA-0.5,and PGS-M-n-HA-0.6 series scaffolds increase with the increase of n-HA content.The proliferation ability of PGS-M-n-HA-0.4,PGS-M-n-HA-0.5,and PGS-M-n-HA-0.6 series scaffolds compared with PGS-M increased after incubation with AD-MSCs.Compared with PGS-M,PGS-M-n-HA series scaffolds promoted the expression of osteogenic differentiation genes:Runx-2?Osteocalcin and Collagen I.Compared with PGS-M,PGS-M-n-HA series scaffolds inhibit the release IL-6.Compared with PGS-M,PGS-M-n-HA series scaffolds had more calcium and phosphorus precipitation after immersion in simulated body fluids.Compared with PGS-M,The results of three dimensional CT and histological examination of the rat skull defect model showed that the PGS-M-n-HA-0.6 scaffold has stronger ability to promote bone formation in vivo.Conclusion:PGS-M-n-HA-0.4,PGS-M-n-HA-0.5,PGS-M-n-HA-0.6series composite scaffolds were successfully prepared by high temperature melting method.The size of pore and the porosity of the PGS-M-n-HA series composite scaffolds are large which may facilitate cell migration and adhesion.The PGS-M-n-HA series composite scaffolds are in a high elastic state at room temperature and body temperature,showing good elasticity and good thermal stability.The degradation of PGS-M-n-HA series scaffolds showed a linear relationship,be good for the growth of the new bone,With the increasing of the proportion of n-HA,the compressive strength and modulus of elasticity of scaffold were increased.The PGS-M-n-HA series scaffolds promote AD-MSCs proliferation and osteogenic differentiation,and decrease inflammatory response.PGS-M-n-HA-0.6 scaffolds promote osteogenesis in vivo.