Study On The Preparation And Biocompatibility Of Hybrid Carbon Nanofiber Bionic Bone Material

Objective:Carbon nanofiber（CNF）materials have been widely used in the field of biomaterials due to their high strength,low density,stable chemical properties,and good biocompatibility.Carbon nanofiber materials also have the advantages of large elastic modulus,large surface area,and almost no reaction with the surrounding tissues of the implanted part in the implanted organism.However,after being implanted into the body as an implant,carbon nanofibers are inert and hardly react with surrounding tissues.After carbon nanofibers are implanted in the body as an implant material,in order to make the carbon nanofiber material better integrate with the implanted part,it is an effective method to combine carbon nanofibers with other materials to make composite materials.The biocompatibility of calcium phosphate bioceramics is excellent.Calcium phosphate bioceramics include hydroxyapatite（HA）,amorphous calcium phosphate（ACP）,tricalcium phosphate（TCP）,calcium pyrophosphate（CPPD）,calcium permeable apatite（DCPD）,tetracalcium phosphate（TTCP）)and so on.Among them,HA and TCP are the most common TCP is similar to HA,and its osteoconductivity and biocompatibility are good.At the same time,the degradation rate of TCP in the body is faster than that of HA.After TCP is implanted into the bone defect,it can be completely degraded in only 6-12 months,so as to be replaced by new bone tissue,and the calcium ions and phosphate ions produced during the degradation of TCP can be Provide raw materials for the mineralization process of new bone tissue From the perspective of bionics,there are a small amount of Zn²⁺and Mg²⁺ in the bone tissue itself,and the introduction of a small amount of inorganic ions to improve the biological activity of the material is an effective method.Magnesium is essential for bone metabolism,can stimulate the formation of new bone,and can also interact with the integrins of osteoblasts,which are responsible for cell adhesion and stability.Mg²⁺ ions released from biological materials can enhance the proliferation and differentiation of osteoblasts,thereby promoting bone regeneration Zinc is a trace element necessary for cells and tissues to maintain normal shape and function.Zinc plays an important role in bone metabolism,and zinc can enhance the osteogenic and mineralization effects of osteoblasts,thereby facilitating the fixation of implants.Zinc supplementation can prevent and treat osteoporosis Doping Mg²⁺and Zn²⁺into carbon nanofibers and β-TCP composite materials can enhance their biocompatibility and promote the formation of bony connections between the composite materials and bone defects after implantation. Methods:（1）Preparation of hybrid carbon nanofiber biomimetic bone material①Hydrolysis of triethyl phosphate（TEP）:add anhydrous ethanol,TEP,and deionized water to the beaker in the ratio of 3mol:1mol:3mol,seal the mouth of the beaker,and continue stirring with a magnetic stirrer at room temperature for 24 hours to mix well.Add ammonia water as a catalyst to the mixed solution,seal the mouth of the bottle,place it in a constant temperature heating magnetic stirrer,set 75℃,and continue magnetic stirring for 24 hours to accelerate the TEP hydrolysis process,and finally obtain a fully hydrolyzed mixed solution②Sol preparation:add Ca²⁺,Zn²⁺,Mg²⁺alcohol solutions to the fully hydrolyzed mixed solution separately,mix the bath with ice and water at 4℃,and continue magnetic stirring to ensure that the solution is fully mixed.After the addition is complete,continue to magnetically stir for 2 hours in an ice-water mixed bath at 4℃.After standing for 7 days,a fully reacted sol was obtained③Electrospinning solution:According to the ratio of DMF:sol=20:1,add 1mL sol to20 mL DMF,then add 10%（w/v）PAN,continue magnetic stirring for 24 hours,and finally make uniform spinning liquid④Electrospinning:The parameters of the electrospinning machine are set as:static voltage 18 kV,receiving distance 15 cm,drum receiver speed 500 RPM,injection pump flow rate 0.6mL/h,temperature 25℃,humidity 35%-45%,each sheet The membranes were collected for 12 hours.After each membrane was taken off,it was dried overnight in a blast electric drying oven at 50℃ to prepare hybrid carbon nanofiber precursors⑤Pre-oxidation of hybrid carbon nanofibers:put the hybrid carbon nanofibers in the burning boat,and then put the burning boat in the tube furnace,set the heating rate to 1℃/min,increase to 270℃,maintain 270℃ for 90 min,And then let it cool to room temperature naturally.⑥Carbonization of pre-oxidized carbon nanofibers:The pre-oxidized carbon nanofibers are placed in a tube furnace,and nitrogen is blown at room temperature for 10 minutes,so that they are in a nitrogen atmosphere,and then heated to 1100℃ at a rate of 5℃/min,and maintained at 1100℃ for 3h,And then it is cooled to room temperature to obtain the final product hybrid carbon nanofiber bionic bone material（2）Characterization of the hybrid carbon nanofiber biomimetic bone material:The hybrid carbon nanofiber biomimetic bone material was characterized by SEM（3）Evaluation of biocompatibility of hybrid carbon nanofiber biomimetic bone material①The MC3T3-E1 cells were seeded on the surface of hybrid carbon nanofibers by cell culture technology,fixed by glutaraldehyde,dehydrated with gradient ethanol,and sprayed with gold.SEM was used to observe the morphology of cell adhesion and proliferation on the surface of hybrid carbon nanofibers②Detect the proliferation and adhesion of MC3T3-E1 cells by MTT method Results:（1）The hybrid carbon nanofiber biomimetic bone material prepared by the combination of gel-sol method and electrospinning technology has stable morphology and diameter distribution,and the content of Zn²⁺and Mg²⁺doped in the material is consistent with expected（2）The hybrid carbon nanofiber biomimetic bone material has good biocompatibility（3）The doping of Zn²⁺and Mg²⁺in the hybrid carbon nanofiber biomimetic bone material can promote the proliferation and adhesion of MC3T3-E1 cells.At the same time,the doping of Zn²⁺is more conducive to the proliferation of osteoblasts than the doping of Mg²⁺.Adhesion,the difference was statistically significant,P<0.05（4）For the promotion of MC3T3-E1 cell proliferation and adhesion,the co-doping effect of Zn²⁺and Mg²⁺is better than that of single Zn²⁺or Mg²⁺.The statistical difference is compared,P<0.05.Conclusion:Our research group combined the gel-sol method with electrospinning technology to prepare a hybrid carbon nanofiber bionic bone material with stable morphology.The doped Zn²⁺and Mg²⁺ in the hybrid carbon nanofiber biomimetic bone material can improve the biocompatibility of the material.Compared with single doping of Zn²⁺ or Mg²⁺,the simultaneous doping of Zn²⁺and Mg²⁺can further improve the biocompatibility of the hybrid carbon nanofiber biomimetic bone material.Biocompatibility of fiber bionic bone materials.This suggests that the hybrid carbon nanofiber biomimetic bone material is expected to become a clinical candidate artificial bone material with good biocompatibility.