In Vivo Degradation Characterization Of Cross-linked Sodium Hyaluronate Gel Based On X-ray Microscopy

Cross-linked sodium hyaluronate products are the first choice in the beauty and plastic industry due to their strong moisturizing and filling properties.The standard YY/T0962-2021《Cross-linked sodium hyaluronate gel for plastic surgery》stipulated the detection methods of the product in terms of appearance,infrared identification,protein content,sodium hyaluronate content and biological evaluation.The in vivo degradation of cross-linked sodium hyaluronate gel can only be evaluated qualitatively by HE staining.In this paper,X-ray microscopy will be used to explore the quantitative analysis of the degradation of cross-linked sodium hyaluronate in vivo,to explore the feasibility of X-ray microanalysis method in quantitative evaluation of the degradation of cross-linked sodium hyaluronate in vivo,and to verify the method through biosafety evaluation.The main research results are as follows:（1）Three-dimensional X-ray microscopy can observe the internal three-dimensional structure of cross-linked sodium hyaluronate gel materials.High-quality images can be obtained by optimizing imaging parameters.The imaging parameters of cross-linked sodium hyaluronate gel implanted in rabbits at different time points were optimized,and the parameters were determined as follows:XRM scanning parameters of samples implanted for 2 weeks:source setting 60 kV,power 5.02 W,exposure time 5.0 s,source filter LE 2,Binning=1,objective 0.4 X,pixel size 5.59μm.XRM scanning parameters of samples implanted for 4 weeks:source setting 60 kV,power 3.42 W,exposure time 4.0 s,source filter LE 2,Binning=1,objective 0.4 X,pixel size 5.03μm.XRM scanning parameters of samples implanted for 8 weeks:source setting 60 kV,power 4.97 W,exposure time 5.0 s,source filter LE 2,Binning=1,objective lens 0.4 X,pixel size 10.40μm.XRM scanning parameters of samples implanted for 12 weeks:source setting 60 kV,power 4.97 W,exposure time 2.0 s,source filter LE 2,Binning=2,objective 0.4 X,pixel size 15.85μm.XRM scanning parameters of samples implanted for 26 weeks:source setting 60 kV,power 5.00 W,exposure time 4.5 s,source filter LE 2,Binning=1,objective lens 0.4 X,pixel size 8.39μm.（2）Quantification of degradation of cross-linked sodium hyaluronate gel in vivo requires the use of software.This paper briefly introduces the process of quantitative analysis of in vivo degradation of cross-linked sodium hyaluronate gel by using software tools,and uses"deep learning"commands to segment cells and materials and calculate the volume of materials.（3）X-ray microanalysis was used to study the degradation of cross-linked sodium hyaluronate in vivo.After 2 weeks of implantation of cross-linked sodium hyaluronate gel in rabbits,the remaining material of cross-linked sodium hyaluronate was 2.83×10¹⁰μm³.After 4 weeks of implantation in rabbits,the remaining cross-linked sodium hyaluronate material was 1.11×10¹⁰μm³;After 8 weeks of implantation in rabbits,the remaining cross-linked sodium hyaluronate material was 6.15×10¹⁰μm³;After 12 weeks of implantation in rabbits,the remaining cross-linked sodium hyaluronate material was8.40×10¹⁰μm³;26 weeks after implantation in rabbits,the remaining cross-linked sodium hyaluronate material was 3.43×10¹⁰μm³.（4）The in vivo degradation method of cross-linked sodium hyaluronate gel by X-ray microscopy was verified by biological evaluation.After the cross-linked sodium hyaluronate gel was implanted into rabbits for a period of time,HE staining was performed on the implantation site.Combined with the result of HE staining,fiber tissue in the gel grew and the gel was divided into several small sacs of different shapes and sizes,indicating that the quantitative degradation of the gel in vivo by X-ray microscopy was feasible.Combined with the results of immunohistochemical staining,the positive expression of MMP-9 at the implantation site decreased first and then increased after the gel was implanted in rabbits,indicating that the quantitative degradation of the gel in vivo by X-ray microscopy is feasible.The expression of IL-6 in rabbit serum was detected by ELISA.The results showed that no IL-6 expression was detected in rabbit arterial blood serum.Blood biochemical results showed that after gel implantation in rabbits,ALP increased significantly at the initial stage of implantation,and then slowly returned to normal level,and other blood biochemical indexes were within the normal reference range.These results indicated that the determination of IL-6 expression in serum and blood biochemistry could not verify the determination of gel volume by X-ray microscopy.