Preparation And Biological Evaluation Of Amorphous Calcium Polyphosphate Coatings With Different Chain Lengths On Pure Titanium

ObjectiveBone defects have plagued orthopaedic surgeons for centuries,and normal bone tissue is unable to bridge and repair large segments of bone defects（bone defects longer than 2-2.5times the diameter of the affected bone）.Large bone defects caused by trauma,tumors,infections,and other diseases of the locomotor system seriously affect the quality of life of millions of people each year.The metal titanium and titanium alloys have long been used as the material of choice for orthopedic grafts.However,titanium itself is biologically inert and has a poor osteogenic capacity,which slows down the postoperative recovery time.In addition,the need for better intraoperative and postoperative hemostasis of grafts has also been a major concern in recent years.Therefore,research has been devoted to the surface modification of titanium implants and the establishment of various bioactive coatings to achieve better osseointegration and hemostasis.Inorganic polyphosphates（poly P）,a compound with similar composition to bone minerals,have attracted the attention of researchers as a polyanion with a strong negative charge that can combine with divalent and trivalent metal cations to play different roles,especially in osteogenic reactions and in regulating the coagulation process.It is known that the process of bone mineralization requires large amounts of Ca²⁺,so many researchers have tried to combine poly P with Ca²⁺to form amorphous calcium polyphosphates（Ca-poly P）.Inspired by this,our group tried to prepare Ca-poly P coating on the surface of titanium metal.By using different chain lengths of poly P and calcium chloride dihydrate as raw materials,Ca-poly P was grown in situ on the surface of titanium sheets to form a coating by the principle of charge attraction,and its biological effects in vitro were investigated in order to make the modified titanium not only have excellent osteogenic ability but also have certain coagulation ability.Methods:1.After applying sandpaper to polish the pure titanium surface,the titanium flakes were immersed in poly P solutions of different chain lengths by the principle of charge attraction,followed by the dropwise addition of supersaturated calcium chloride dihydrate solution under the condition of p H=10 and waiting for 24 h.The experimental groups were:polished titanium flakes loaded with Ca-poly P coatings of 3,6 and 20 chain lengths,noted as Ca-P3-Ti,Ca P6-Ti and Ca-P20-Ti,respectively;the control group was for for polished titanium flakes after grinding,noted as Ti.2.The surface morphology and microstructure of the samples were studied by field emission scanning electron microscopy（SEM）,the chemical composition and elemental content of the surface coating of the samples were analyzed by X-ray energy spectrometry（EDS）,the material structure was analyzed by X-ray diffractometer（XRD）,the samples were chemically analyzed by Fourier infrared spectroscopy（FTIR）,and the hydrophobicity of the specimens was measured by contact angle measurement.The basic characterization of the material was completed by using a contact angle meter.3.The experimental and control groups were prepared,and mouse embryonic osteoblast precursor cells（MC3T3-E1）were inoculated on the surface of the material,and the cell culture and CCK-8 cytotoxicity tests were completed using enzyme markers and other instruments to demonstrate the in vitro cytocompatibility and cell proliferation ability of the modified titanium flakes.4.The modified titanium sheet and the smooth pure titanium sheet were prepared,placed in24-well plates,and MC3T3-E1 cells were inoculated on the surface of different specimens,and the quantitative alkaline phosphatase activity assay,alkaline phosphatase staining assay and Real-time fluorescence quantitative PCR（RT-PCR）assay were completed using enzyme standard,fluorescence inverted microscope and PCR thermocycler,respectively,so as to complete the cell osteogenic differentiation-related The osteointegration ability of the surface coating of titanium flakes was demonstrated.5.The experimental and control groups were prepared and placed in 24-well plates,and MC3T3-E1 cells were inoculated on the surface of the material,and the quantitative protein adhesion assay and the concentration of rat platelet-derived growth factor AB（PDGF-AB）and rat transforming growth factorβ1（TGF-β1）during the in vitro coagulation process were measured by using enzyme marker and fluorescence inverted microscope,respectively,to demonstrate the coagulation ability of the titanium sheet surface coating.Results:1.Material characterization results:SEM showed that Ca-poly P coatings of 3,6 and 20 chain lengths existed as amorphous structures on the surface of titanium sheets;EDS showed that the closely bound amorphous material on the surface of titanium sheets in each experimental group mainly consisted of calcium,oxygen and phosphorus elements;XRD showed that the peak positions of several groups of specimens were basically consistent with the standard spectrum of titanium,indicating that the material surface coatings were amorphous products rather than crystalline states;The FTIR analysis showed that the peaks of each chain length coating group corresponded to the corresponding chemical bonds;the contact angle measurement showed that the specimens in each experimental group showed significant hydrophilicity compared with the control group.2.Cell culture and cell proliferation results:the cultured MC3T3-E1 cells were in good condition.In the CCK-8 experiment,Ca-P3-Ti,Ca-P6-Ti and Ca-P20-Ti showed good cytocompatibility and better cell proliferation promotion than pure titanium group.Among them,Ca-P20-Ti had the best ability to promote cell proliferation.3.In vitro osseointegration ability assay results:Ca-P3-Ti,Ca-P6-Ti and Ca-P20-Ti all demonstrated better osseointegration ability than pure titanium group.The longer the chain length of Ca-poly P coating,the better the ability to promote osteogenic differentiation.Among them,the Ca-poly P coating with 20 chain length had the best ability to promote osteointegration.4.In vitro coagulation assay results:Ca-P3-Ti,Ca-P6-Ti and Ca-P20-Ti showed better coagulation effect than the pure titanium sheet group.the longer the chain length of Ca-poly P coating,the better the protein adhesion on the surface of the titanium sheet and the greater the concentration of platelet growth factor released from the clot.The Ca-poly P coating with 20 chain length had the best procoagulant ability.Conclusion:The modified titanium sheets coated with different chain lengths of Ca-poly P prepared by our group had better capacity for promoting cellular proliferation,osseointegration,and coagulation than pure titanium,and the longer the chain length of Ca-poly P the better the biological activity.Among them,the coating of 20-chain-length Ca-poly P was the most effective.Significance:In this study,the bioactivity of pure titanium implants was substantially improved by an extremely simple preparation method,allowing the inert titanium metal to acquire both osteogenic and pro-coagulant abilities.It provides new ideas for the construction of implant coatings with excellent biological efficacy.If used in clinical work in the future,it can speed up the recovery of patients,accelerate postoperative coagulation and new bone crawling,obtain stronger support at an early stage,make the force transmission more physiological,and promote early mobility to avoid more postoperative complications.