Mechanism Of Accelerated Callus Formation In Moderate-severe Traumatic Brain Injury Combined With Femur Fracture

Objectives: Bone disunion and delayed healing of fracture would seriously affect the fracture healing and subsequent quality of life of patients with fracture,which was mainly manifested as failure to heal normally beyond normal time and failure to connect normally at the fracture site due to poor callus growth.At present,the specific mechanisms of delayed healing in patients with bone disunion fracture were not fully understood and explored,and there was a lack of effective therapeutic drugs,leading to serious limitations in research progress.A large number of studies have shown that the phenomenon of accelerated callus formation could be observed in patients with traumatic brain injury combined with fracture compared with patients with normal simple fractures.However,as for the mechanism,existing studies mainly focused on the presence of substances in the serum of patients with traumatic brain injury combined with fracture or animal models that could accelerate callus formation at the fracture site,such as neurotrophic factors and inflammatory mediators,etc.But it still didn’t fully explain the phenomenon.The main reason for this phenomenon was that the researchers was failed to consider the causes of the phenomenon as a whole body.By exploring the mechanism of accelerated callus formation at the fracture site after traumatic brain injury combined with fracture,this study provided a new therapeutic direction for patients with delayed union of bone disunion after fracture.In addition,behavioral and neurobiochemical studies were conducted to investigate the functional changes of the glymphatic system in mice and the role of riboflavin in accelerated callus formation after traumatic brain injury and fracture.Methods:By constructing traumatic brain injury model mice,closed femur fracture model mice,traumatic brain injury combined with fracture model mice,combined with neurofunctional scores,and Micro-CT,we analyzed the local callus formation of fractures in mice with moderate to severe traumatic brain injury combined with fracture.The open field experiment,rotating rod experiment,and pole test were reused to explore the motor function recovery of mice with moderate and severe traumatic brain injury combined with fracture compared to simple fracture mice.Subsequently,injection of cerebellar medulla cisterna was used to explore the possible pathway of relevant regulatory substances transport and the functional changes of the glymphatic system after moderate to severe traumatic brain injury combined with fracture.Untargeted metabolomics was used to analyze the differences in serum and sciatic nerve substances in mice with moderate to severe traumatic brain injury combined with fracture,and to identify the key metabolites that accelerate callus formation.Subsequently,the standard substance of riboflavin was analyzed by gas-liquid mass spectrometry,and the standard curve was drawn for the following study.Then,the differences of riboflavin contents in the serum,brain tissue,spinal cord tissue and sciatic nerve tissue of mice with moderate to severe traumatic brain injury combined with fracture and simple fracture were quantitatively analyzed by gas-liquid mass spectrometry,and the possible transport route of riboflavin was identified.Meanwhile,the effects of riboflavin at different concentrations on osteoblast proliferation were investigated by cell proliferation assay,and the effects of riboflavin on proliferation,differentiation and migration of osteoblasts were investigated by western blotting assay,real-time quantitative PCR,and wound healing analysis.Finally,the closed femur fracture model mice were constructed,and normal saline and the corresponding concentration of riboflavin were injected respectively to explore the influence of riboflavin on the recovery of motor function after fracture in the same period.Results: The results of Micro-CT showed that callus formation was accelerated in mice with moderate to severe traumatic brain injury combined with fracture compared with mice with simple fracture,and bone density,callus volume,and bone volume fraction increased significantly at the same time.Meanwhile,behavioral experiments showed that the total motion distance of mice with moderate to severe traumatic brain injury combined with fracture increased significantly compared with mice with a simple fracture in open field test.In the rotating rod experiment,the stay time was obviously prolonged,and in the pole test,the downtime of mice with the injection of riboflavin was obviously shortened.The experimental results of cerebellar bulbar cisterna injection showed that the fluorescence intensity in the brain of mice with moderate and severe traumatic brain injury combined with fracture was significantly decreased compared to mice with simple fracture,and the fluorescence intensity of spinal cord and sciatic nerve was significantly enhanced in mice with simple fracture.Non-targeted metabolomics results showed that riboflavin in serum samples of mice with moderate to severe traumatic brain injury combined with fracture was not significantly increased compared with mice with simple fracture,and differentially expressed metabolites were not enriched into riboflavin-related metabolic pathways,while in sciatic nerve samples,riboflavin was significantly increased in mice with moderate to severe traumatic brain injury combined with fracture compared with mice with simple fracture.Pathway enrichment analysis showed that riboflavin metabolism played a key role.Quantitative analysis by gas-liquid mass spectrum showed that the serum riboflavin content of mice with moderate to severe traumatic brain injury combined with fracture was not significantly different from that of mice with simple fracture.The content of riboflavin in brain tissue was significantly decreased,the content of riboflavin in spinal cord was significantly increased,and the content of riboflavin in sciatic nerve was significantly increased.In the verification experiment,the total movement distance of mice in the fracture + riboflavin injection group was significantly increased compared with that in the simple fracture group,the stay time of mice in the rotating rod test was significantly extended,and the time of mice in the pole test was significantly shortened.In cell experiments,riboflavin can significantly promote the proliferation and differentiation of osteoblasts,and the concentration of 10μM is the most significant effect,and riboflavin can up-regulate the expressions of PCNA,RUNX2 and ALP in osteoblasts,and significantly promote the migration and healing capacity of osteoblasts.Conclusion: After moderate to severe traumatic brain injury combined with peripheral fracture,riboflavin can significantly accelerate callus formation and motor function recovery.After moderate to severe traumatic brain injury combined with peripheral fracture,riboflavin can reach the fracture site through the brain-spinal-sciatic nerve pathway,nourish local osteoblasts,promote the proliferation and differentiation of local osteoblasts and enhance the migration ability of osteoblasts,thus promoting the recovery of motor function of the affected limb.