The Preparation, Characterization And Bioactivity Evaluation Of Composite Material Containing Strontium And PRF In Vitro

Implant denture is regarded as “the third set of teeth” and has more irreplaceableadvantages than traditional dental prosthesis. But some dentition defect cases can’tachieve the initial stability of the implant since they have the big problem of bonedefect, and it limits the clinical application of implant. Bone graft is the primarymethod to solve this problem. But autografts and allografts have their limitations.These include the need for a second site of surgery, limited availability of appropriatetissue, and/or risk of immunogenicity. At present, the researchers are focusing ongaining ideal synthetic bone scaffold materials, which provide support for the growthof new bone and occupy an important position in bone tissue engineering. Butpolymer scaffold materials have some limitations, like lower biological activity, poormechanical properties, and easy to cause aseptic inflammation. Therefore, themethod of increase the biological activity and promote the osteoblast cell proliferationis the main purposes of research at present.Strontium, atomic number38, it is one of the trace elements in human body, andwas isolated by Humphry Davy in1808. Studies have shown that Strontium has adouble effect on bone metabolism--increasing new bone formation and reducing boneresorption. Strontium is widely used as a systemic treatment of osteoporosis at present,but its lack of targeting limits its application. At present, the researchers are focusingon the local application of strontium in bone reconstruct.Platelet-rich fibrin（PRF）was first described by Choukroun. It does not requireany agents during preparation, so it may be considered as a autografts. Past work hasshown that PRF concentrates the growth factors, which have been identified as key players in the process of tissue remodeling. Currently, PRF is widely used in thetreatment of cartilage defects, soft tissue defects, and bone defects. However, it mustbe used immediately after preparation, the time of growth factor release process is tooshort to improve the bone reconstruction process and its poor mechanical strengthhave limited its application in bone tissue engineering. Therefore, a suitable method tosolve these problems is the hot research topic at present.For years, hydrogel has been used as a controlled and sustained-release system.Its general characteristics resemble those of a native extracellular matrix. Thetemperature-sensitive triblock copolymer poly-(D,L-lactic acid-co-glycolic acid)(PLGA)-polyethylene glycol (PEG)-PLGA (PLGA-PEG-PLGA) was first reported byZentner (2001). This copolymer exhibits a reversible sol–gel transition and thecross-linking can occur over a wide range of temperatures, including those that arephysiologically relevant (37oC). However, as with PRF, its application for use in bonedefects has been limited due to its poor mechanical strength.Could PRF and strontium be loaded into the scaffold through hydrogel andwhether the composite material can slow release the drug and promote bone formationis uncertain from the report of domestic and international researchers.In our research, we evaluated the feasibility and the biosafety of the scaffoldcombine with the hydrogel. We evaluated the control release effect of the compositescaffold. We evaluated the effect of composite material on the adhesion, proliferationand differentiation of MG63cell in vitro. We used real time PCR to detect compositematerial on bone formation related genes. And then, their influence on Collagen I,Runx2and OPN protein was detected by Western Blot. Our results provide theoreticalbasis for clinical application in the future.1.The influence of different concentration of SrCl2on the proliferation anddifferentiation of MG63cell. The results show that, SrCl2has the strongest ability topromote MG63proliferation when the concentration is500μg/ml. Thus, we choosethis concentration in our following study.2.Evaluated the feasibility of lyophilized PRF and find the method to obtain the concentration of growth factors with scanning electron microscope (SEM)and enzyme linked immunosorbent assay (ELISA). The results show that, thelyophilized PRF is more porous than PRF, and the fibers are thinner than PRF. Thelyophilized PRF powder had a burst released of growth factors initially, thus we mayobtain the growth factors concentration solution.3.We evaluated the feasibility of the scaffold combine with the hydrogel. Weevaluated the preparation, characterization and degradation of the compositescaffold in vitro. The results show that, we successfully prepared thePLGA-PEG-PLGA hydrogel, and the cross-link temperature is34oC. The nHA/PLGA scaffold has the typical polymerizer poriform structure with the pore size of150-270μm and the pores were interconnected. The hydrogel successfully entered thepores of the scaffold, the pores were not blocked by the hydrogel, and that thenHA/PLGA/Gel scaffold still contained interconnected and irregular pores. Theaverage pore diameter of nHA/PLGA/Gel was70-160μm, suitable structure for bonetissue engineering. Inductively coupled plasma mass spectrometry(ICP-MS) andenzyme linked immunosorbent assay(ELISA) show that, the release of strontium fromcomposite material was32.2%in the first week and shown a linearity slow release in2-4weeks, then reach the total release amount of70.3%in the12thweek, the releaseof growth factors of PRF from composite material was34.03%in the first week andshown a linearity slow release in2-4weeks, then reach the total release amount of76.5%in the12thweek. The degradation of nHA/PLGA/Gel scaffold material wasslightly faster in the first three weeks and then reaches a linearity mode by in vitroweight loss testing. The degradation of nHA/PLGA/Gel scaffold material was13.4%at the end of12weeks while the weight loss of nHA/PLGA scaffold material was8.2%. The PH of incubation buffer for nHA/PLGA/Gel scaffold material was6.93±0.1and for nHA/PLGA scaffold material was6.99±0.1at the end of12weeks.The compress experiment shown the compressive strength of the scaffold loaded withhydrogel has been improved significantly. The compressive strength of nHA/PLGAscaffold material was (2.31±0.18)MPa and the compressive strength of nHA/PLGA/Gel scaffold material was (2.90±0.16) MPa.4.We evaluated the biological safety of the composite scaffold and theinfluence of drug-loading composite material on the adhesion, proliferation anddifferentiation of MG-63cell. The results show that, the PLGA/nHA/CMs scaffoldwas safe to be used and had no acute hemolytic activity. The drug-loading compositematerial could promote the adhesion, proliferation and differentiation ofosteoblast-like cells.5.The influence of drug-loading composite material on the related functionalgene and protein of MG63cell.The results of Real-time PCR showed that the expression of CollagenⅠmRNAin MG63cells in experimental group1was no significant difference with controlgroups in the1stday, the expression of CollagenⅠmRNA was significantly higher inthe experimental group1than the control groups in the3rdday, expression in theexperimental group2was higher than the scaffold control group, the expression ofCollagenⅠmRNA was significantly higher in the experimental group1than othergroups in the5thday. The expression of OPN mRNA in MG63cells in experimentalgroup, the experimental group2and the scaffold control group were significantlyhigher than the blank control group in the1stday, the mRNA expression inexperimental group1and the experimental group2were significantly higher than theother groups and the experimental group1is higher than the experimental group2inthe3rdday, the mRNA expression in experimental group1and the experimentalgroup2were significantly higher than other groups in the5thday. The expression ofRunx2mRNA in MG63cells in experimental group1was significantly higher thanother groups in the1stday, the mRNA expression in experimental group1and theexperimental group2were significantly higher than the other groups and theexperimental group1is higher than the experimental group2in the3rdday, the mRNAexpression in experimental group1and the experimental group2were significantlyhigher than the other groups in the5thday. The expression of SP7mRNA in MG63cells in experimental group1was no significant difference with control groups in the 1stday, the mRNA expression in experimental group1, experimental group2andscaffold control group were significantly higher than blank control group in the3rdday, the mRNA expression in experimental group1and the experimental group2weresignificantly higher than the other groups in the5thday.The results of Western Blot showed that the expression of Runx2protein wasobviously higher in experimental group1and blank control group than the other twogroups in the1stday, the protein was obviously higher in scaffold control group andblank control group than the other two groups in the3rdday, the protein was obviouslyhigher in experimental group1and experimental group2than the other two groups inthe5thday. The expression of OPN protein was obviously higher in experimentalgroup1and experimental group2than the other two groups and the experimentalgroup1is higher than the experimental group2in the1stday, the protein expression inexperimental group1, experimental group2and scaffold control group were obviouslyhigher than the blank control group in the3rdday, the protein was obviously higher inexperimental group1and blank control group than the other two groups and theexperimental group1was higher than the blank control group in the5thday. Theexpression of CollagenⅠprotein was obviously higher in scaffold control group thanthe other groups in the1stday, the protein expression in experimental group1andscaffold control group were obviously higher than the experimental group2and blankcontrol group, the experimental group1was higher than the scaffold control group, theexperimental group2was higher than the blank control group in the3rdday, theprotein was obviously higher in experimental group1than the other groups, theexperimental group2and scaffold control group were higher than the blank controlgroup in the5thday.In conclusion, SrCl2has a noticeable promoting effect on the proliferation anddifferentiation of osteoblast cell. The ground lyophilized PRF is an effective methodto obtain the growth factors concentration. The drug-loading hydrogel is successfullyentered the scaffold and slow release the growth factors without destroy the scaffoldstructure. The nHA/PLGA/Gel has a well biological safety and a well mechanical strength.The nHA/PLGA/Gel/Sr/PRF composite material has a noticeable promotingeffect on the adhesion, proliferation and differentiation of osteoblast cell and canpromote the bone formation related gene and protein expression in the early stage.This research provide theoretical basis for the dental implant clinical application ofcontrolled delivery of Sr and growth factors of PRF composite material.