| Pathological calcification in oral craniomaxillofacial soft tissues can be caused by trauma,infection and joint replacement.This type of pathological calcification in soft tissues can cause dysfunction such as pain in the jaw and face,joint stiffness,and limited motion in patients.One of the more serious types of pathological calcification,heterotopic ossification,causes abnormal calcified tissue,or even mature bone-like tissue,to appear in soft tissues where calcification should not occur.These pathological mineralized crystals can cause continuously aggravated pain to patients at least and even endanger their lives in severe cases because heterotopic ossification often increases in size as the disease progresses.Because heterotopic ossification squeezes the adjacent tissues,it may cause repeated pain to the patient or endanger the patient’s life.The development of HO is causally related after significant initial local tissue injury.It should be noted that casualties of traffic accidents and military operations are prone to heterotopic ossification,even as high as 50%,which is significantly higher than the population prevalence of this disease.Analysis of the reasons for this reveals that injured patients in both settings often have traumatic brain injuries along with peripheral injuries.In order to distinguish these heterotopic ossifications,the researchers named these heterotopic ossifications in combination with traumatic brain injury(TBI)as neurogenic heterotopic ossifications(NHO).Current treatment options for NHO are mainly limited to symptomatic surgical therapy and drug therapy.However,the above treatments have the disadvantages of drug side effects and high recurrence rates after operation.The reason for this is that the mechanisms underlying the onset and development of NHO have not been well studied.In recent years,extracellular vesicles(EVs)have been found to play an important role in intercellular communication and interaction by carrying biological information such as proteins and nucleic acids.One study confirmed that the percentage of NEV in peripheral blood is significantly higher in patients after the onset of TBI.It has been further shown that NEV can carry inflammatory proteins such as IL-1β,HMGB1,etc.After intake of these proteins,damage and destruction occur in the local tissue.These studies suggest that the role of NEV in regulating the microenvironment within peripheral tissues deserves further exploration.Our previous studies have shown that under the stimulation of inflammatory factors,endoplasmic reticulum stress and mitochondrial dysfunction will occur in cells.Because both organelles are important sites for calcium and phosphorus metabolism in the cell,their dysfunction may lead to an increased concentration of calcium and phosphorus in organelles,resulting in the formation of amorphous calcified precursors(ACPs).With the occurrence of cell death,these ACPs can be released into extracellular matrix,which may participate in the subsequent development of HO.Besides,it has been reported that after cell death,apoptosis or necrosis,the amounts of mineralized materials(including local nucleic acid or pyrophosphate)are increased remarkably,further leading to tissue calcification.Then,how NEV after TBI is related to the occurrence of NHO will be an important scientific question in this study.MethodsIn the first part of the experiment,we constructed rat models of neurogenic heterotopic ossification by combining the trauma-induced tendon HO with electric hammer-induced TBI.Then we used Micro-CT,H&E staining and other morphological characterization methods to compare the effect of TBI on HO.In the second part of the experiment,we investigated whether NEVs participate in the development of NHO by immunofluorescence,injection of NEVs and secretion blocking experiments.We studied the contents of NEVs by using mass spectrometry and found that NEV has a role in inducing pyroptosis.Finally,we investigated the presence of pyroptosis in the traumatized Achilles tendon of NHO rats by means of Westernblot,TEM,immunofluorescence,immunohistochemistry and other techniques,and further detected the types of pyroptosis cells by immunofluorescence.In the third part of the experiment,we co-cultured the NEV with the identified target cells.To clarify whether NEV could induce pyroptosis of target cells,we observed the target cells by immunofluorescence,TEM,Westernblot and other techniques in vitro after coculture.We also verified the changes in calcium and phosphorus concentrations after pyroptosis in vitro.In the fourth part of the experiment,we infused NEV back into rats,verified the effect of NEV by immunofluorescence,H&E and Micro-CT.We investigated the effect of inhibition of pyroptosis on the development of NHO by pharmacologically inhibiting pyroptosis and again using immunofluorescence,H&E and Micro-CT to detect local pyroptosis and calcification of heterotopic ossification.ResultIn the first part,a comparison between the NHO rat model and the simple peripheral injury(HO)rat model showed that the probability of heterotopic ossification of the Achilles tendon was significantly higher in the NHO rat model(p<0.05),and the Bone Volume/Tissue Volume(BV/TV)and Bone mineral density(BMD)of heterotopic ossification were significantly larger in the NHO rat model than in the HO rat model at 3 and 5 weeks after modeling(p<0.05).The results showed that the arrangement of collagen fibers in the normal Achilles tendon was dense and orderly.The collagen fibers in the traumatized Achilles tendon were broken and disorganized with inflammatory cell infiltration.3 weeks after surgery,small calcified structures appeared locally in the Achilles tendon of the NHO group.At the same time,the Achilles tendon of the HO group was dominated by hyperplastic fibers and no obvious calcified structures were seen.5 weeks after surgery,localized calcification was observed in the Achilles tendon of NHO group rats and bone marrow cavities was observed in the middle of the calcified structure.Rats in the HO group also showed ectopic ossified structures containing bone marrow cavities,but the trabecular thickness(TB.Th)and trabecular number(TB.N)were significantly smaller than those in the NHO group(p<0.05).In the second part of the experiment,the percentage of BV/TV,BMD,TB.Th and TB.N of heterotopic ossification in the Achilles tendon of HO rats injected intravenously with NEV were significantly higher than those in the HO rat model(p<0.05).However,the percentage of BV/TV,BMD,TB.Th and TB.N of heterotopic ossification in the Achilles tendon of NHO rats injected intravenously with extracellular vesicle secretion inhibitors were lower than those in the NHO rat model(p<0.05).Secondly,this study also found that NEV carried HMGB1 protein,which had the effect of inducing cell pyroptosis.The percentage of dead cells localized in Achilles tendon trauma was significantly higher in NHO rats than in HO rats(p<0.05).In addition,the percentage of NLRP3-positive cells and the percentage of Active CASPASE-1-positive cells were significantly higher in NHO rats than those in HO rats with localized Achilles tendon trauma(p<0.05).Notably,this experiment confirmed that the local pyroptosis cells in the Achilles tendon of the NHO rat model were mainly fibroblasts.In the third part of the experiment,three types of extracellular vesicles(EV,NEV and NEV-depleted EV)obtained from the previous isolation were co-cultured with identified fibroblasts.N-GSDMD fluorescence intensity and the area ratio of alizarin red S were significantly higher in the NEV group than that in the(NEV-depleted EV)group(p<0.05).N-GSDMD fluorescence intensity of(NEV+pyroptosis inhibitor)group was lower than that of NEV group(p<0.05).The area ratio of alizarin red S in(NEV+pyroptosis inhibitor)group was lower than that in NEV group after 7 days of co-culture(p<0.05).Calcified nodules in(NEV+pyroptosis inhibitor)group and(NEV-depleted EV)group were significantly less than those in NEV group after 7 days of co-culture(p<0.05).The results of elemental analysis showed that the calcium content of calcified nodules in NEV group was significantly more than those in(NEV+pyroptosis inhibitor)group and(NEV-depleted EV)group.In addition,the calcium and phosphorus content of the culture medium was found to be elevated after pyroptosis of fibroblasts(p<0.05).In the fourth part of the experiment,the content of N-GSDMD protein in local cells of HO rat model was significantly increased after intravenous injection of NEV(p<0.05)and NEV positive cells accounted for more than 70%among N-GSDMD positive cells.In addition,the local calcium and phosphorus content of Achilles tendon trauma was significantly increased(p<0.05).Intravenous injection of pyroptosis inhibitors in NHO rats significantly reduced the number of N-GSDMD positive cells in the local Achilles tendon injury,and significantly reduced the BV/TV and BMD of heterotopic ossification of Achilles tendon(p<0.05).Conclusion1.In this study,we successfully constructed a NHO model in rats.At the same time,localized heterotopic ossification of the Achilles tendon is more severe in the NHO rat model than that in HO rat model.2.NEV plays an important role in the development of NHO.NEV can carry protein HMGB1,which triggers pyroptosis.The number of pyroptosis fibroblast increased significantly in the traumatized Achilles tendon of NHO rats.3.Uptake of NEV can induce the pyroptosis of fibroblasts.The release of calcium and phosphorus after pyroptosis can lead to the formation of tiny calcified crystals.4.NEV injection exacerbated pyroptosis and increased local calcium and phosphorus levels in traumatized Achilles tendon.Blocking pyroptosis alleviated the disease process of NHO.To sum up,we found a possible mechanism of NHO.The injured central nervous system releases NEV into the peripheral circulation,which promoted the pyroptosis of fibroblasts in injured site.The release of calcium and phosphorus after pyroptosis can lead to the formation of tiny calcified crystals,which promotes the formation of NHO. |
PDF Full Text Request