The Role And Mechanism Of MMP13 In Metabolic Imbalance Of ECM In Injured Tendons

BackgroundIn recent years,with the vigorous development of social economy and the increasing improvement of people’s living standards,physical exercise and sports are increasingly popular among the general public.The number of people and types of sports is also gradually increasing,and as a result,the incidence rate and types of sports injuries are also increasing,of which tendon injuries accounting for 50%of them.Currently,the clinical treatment of tendon injury has poor efficacy,and complications such as pain,limited mobility,and re-rupture often occur.This situation seriously affects the quality of life of sports enthusiasts and brings heavy economic and social burdens to both the country and individuals.The treatment of tendon injuries has become a major challenge for orthopedic and sports medicine physicians.Tendons are dense connective tissues with type I collagen（accounting for 80%of dry weight）as their main component.Previous studies have generally believed that the disruption of the anabolism-catabolism homeostasis of the extracellular matrix（ECM）and the increased catabolic activity after injured tendon led to structural damage and reduced mechanical performance of the tendon,which is the main reasons for the current clinical dilemma.The tendon ECM homeostasis and remodeling functions are jointly maintained by endogenous Matrix Metalloproteinase（MMPs）and Tissue inhibitor of matrix metalloproteinases（TIMPs）.MMPs are the dominant catabolic activity of tendon ECM,synthesized and secreted by TDSCs,the stromal cells of tendon tissue.Their main function is proteolytic enzymes that degrade collagen and other structural molecules.There are many driving factors for the increased catabolic activity of ECM after tendon injury,such as inflammatory factors,oxidative stress,mechanical factors,and there is currently no consensus.Researchers do not have a deep understanding of the regulatory mechanism of ECM anabolism-catabolism homeostasis after tendon injury.More research has focused on the expression of MMPs as an indicator of the severity of tendon injury or tendinopathy,so it is currently unable to provide effective interventions for clinical treatment of tendon injury.In summary,we believe that in-depth understanding of the drivers and mechanisms of increased ECM catabolic activity in injured tendons can provide targeted therapeutic strategies for clinical improvement of tendon injury.Objective1.Analysis of the imbalance characteristics of ECM anabolism-catabolism homeostasis in injured tendons;2.Clarify that cell"crowding"after tendon injury promotes MMP13 expression in injured tendons through Piezo1;3.Reveal that ROS signaling mediates the promotion of MMP13 expression by tendon injury and cell"crowding".Methods1.Analysis of homeostasis changes in ECM anabolism-catabolism in the injured tendonsA mouse Achilles tendon transection injury model was established,and the time points after injury were grouped.Samples were collected from the Achilles tendon tissue,and total RNA was extracted for transcriptome sequencing.The changes in the imbalance of ECM anabolism-catabolism homeostasis after tendon injury were analyzed.Then analyze the key proteolytic enzymes involved in ECM catabolic activity.In addition,a mouse transection injury model and a Achilles tendon Punch injury repairing model were established.Tendon tissue at different time points after injury was collected,and total RNA and protein were extracted,and tissue frozen sections were performed.The changes in histology and biochemistry of MMP13 after tendon injury and during the repairing process were evaluated by biochemical methods:RT-PCR（Real time Quantitative Polymerase Chain Reaction）,Western blot,and histological staining:HE staining,Sirius red staining,and immunohistochemistry staining.2.To explore the mechanism of cell"crowding"activating Piezo1 mediated mechanical signal transduction and regulating MMP13 expression in injured tendonsPrimary cells were extracted from mouse tail tendons for culture and passage,and specific markers of TDSCs（Tendon derived stem cells）were identified by fluorescence and induced differentiation staining.TDSCs simulate cell"crowding"in injured tendons in vitro,verifying that cell"crowding"can promote MMP13 expression.Functional gain and loss experiments were conducted through grouping of Piezo1 activators and specific inhibitors,and RT-PCR was used to detect RNA expression in tissues or cells in vitro to verify that cell"crowding"activates Piezo1 mediated mechanical signal transduction and regulates the expression of MMP13 in injured tendons.3.ROS signaling mediates the promotion of MMP13 expression by injured tendons and cell"crowding"In a mouse model of Achilles tendon transection injury and Achilles tendon Punch injury repairing,NAC（N-acetyl-L-cysteine）was injected locally with neutralizing ROS（Reactive oxygen species）products to obtain tendon tissue samples at different time points after injury.RT-PCR,HE staining,Sirius red staining,and immunohistochemistry were used to clarify the mechanism of inhibiting MMP13 expression in injured tendons after neutralizing ROS.At the same time,using H₂O₂（Hydrogen peroxide）to induce oxidative stress in TDSCs in vitro confirmed that ROS promotes the expression of MMP13 in TDSCs.Then,TDSCs were used to simulate the phenomenon of cell"crowding"in vitro,and different antioxidants were administered to group experiments.Cell RNA expression was detected by RT-PCR.Combined with in vivo and in vitro experimental results,it has been demonstrated that cell"crowding"activates Piezo1 and induces ROS to regulate the expression of MMP13 in injured tendons.Results1.Increased ECM catabolic activity in injured tendons,with the most significant increase of MMP13 expressionTranscriptomic analysis showed that the catabolic activity of the injured tendon ECM was enhanced and the homeostasis of anabolism and catabolism was imbalanced.This is the fundamental reason for the destruction of the components and structure of the injured tendon,resulting in a decrease in the biomechanical properties of the tendon and a tendency for the injury to recur.Among them,the increase in the expression of MMP13 was the most significant in the increased catabolic activity of the injured tendon ECM.Through biochemical detection methods and histological observations,it has been shown that the expression of MMP13 continues to increase significantly over time in injured tendons.This indicates that the key proteolytic enzyme that enhances catabolic activity after tendon injury is MMP13.2.Cell"crowding"promotes MMP13 expression in injured tendons through Piezo1In a mouse Achilles tendon Punch injury repairing model,it was observed that there was a phenomenon of cell"crowding"in the injured area with a significant increase in MMP13positive staining.Using TDSCs with different cell density to simulate cell"crowding"in vitro significantly increases MMP13 expression."It suggests that our cells’crowding alters mechanical signal transduction and promotes MMP13 expression.".The model of Achilles tendon Punch injury in mice was established in vivo and the cell"crowding"was simulated using TDSCs with different cell densities in vitro.The functional gain and loss experiments of Piezo1 were conducted,respectively.Experimental results confirm that cell"crowding"in injured tendons mediates an increase in MMP13 expression by activating Piezo1 to alter mechanical signal transduction.3.ROS signaling mediates the promotion of MMP13 expression by tendon injury and cell"crowding"Cell crowding and Piezo1 activation both increase ROS production.ROS is a recognized damage associated molecular patterns（DAMPs）that can activate a series of catabolic pathways.The H₂O₂ induced TDSCs experiment showed that ROS can significantly increase the expression of MMP13 in TDSCs,indicating that ROS can promote the expression of MMP13.The loss function experiment of ROS showed that neutralizing ROS can significantly inhibit the increased expression of MMP13 in injured tendons.In the constructing simulated cell"crowding"experiment with different TDSCs densities in vitro,various types of antioxidants can inhibit the increase in MMP13 expression.Combined with the above experimental results,it is demonstrated that Piezo1 and ROS jointly regulate the expression of MMP13 in injured tendons.4.The increase in osteoclastogenesis and MMP13 secretion during tendon-to-bone insertion growth led to osteolysis at the tendon-to-bone junction,resulting in a decrease in the biomechanical properties of the tendon-to-bone insertionDuring tendon-to-bone insertion growth,the expression of osteoclast specific markers and MMP13 increased significantly over time.The results of osteoclast specific TRAP staining and MMP13 immunohistochemical staining on frozen sections of a mouse Achilles tendon-to-calcaneal junction reconstruction model and their statistical analysis suggested that the osteolysis phenomenon that led to the decline in biomechanical properties of the tendon-to-bone insertion may be caused by osteoclastogenesis and the secretion of MMP13.ConclusionThrough a detailed transcriptomic analysis of the imbalance between extracellular matrix anabolism-catabolism homeostasis in injured tendons,this study identified that the poor prognosis caused by extracellular matrix degradation and biomechanical decline after tendon injury is due to increased catabolic activity represented by a significant increase in MMP13expression.Firstly,the sustained high expression of MMP13 in injured tendons was demonstrated through biochemical and histomorphological methods in different mouse tendon injury models.Subsequently,after observing the phenomenon of cell"crowding"in injured tendons,simulating cell"crowding"experiments in vitro and functional gain and loss in vivo confirmed that the cell"crowding"in injured tendons activated Piezo1 to alter mechanical signal transduction and promote increased MMP13 expression.Finally,in vivo and in vitro ROS neutralization experiments and in vitro ROS induction experiments confirm that ROS signaling mediates the mechanism by which injured tendons and cell"crowding"promote MMP13 expression.