| Part One Effects of ES-PMMA bone cement on expression levels of inflammatory cytokines in rabbit knee replacement modelPolymethylmethacrylate bone cement(PMMA)is an essential implantable material that has been used in widespread clinical orthopedic procedures.It consists of a solid powder and a liquid,which are mixed for use.It has a certain degree of plasticity and adhesion before curing,allowing it to be filled in complex surgical areas,and after curing it acts as a support and adhesive and withstands certain loads.After curing,bone cement plays a supporting and bonding role,and bears a certain load.In the treatment of osteoporotic vertebral compression fractures,PMMA bone cement is frequently employed and the fixation of prostheses in arthroplasty because it is easy to handle,reliable,and does not loosen easily.Studies have demonstrated that bone cement implantation syndrome(BCIS)can occur after bone cement implantation in artificial knee replacements,causing a range of symptoms such as decreased blood pressure,rapid heart rate,and reduced partial pressure of oxygen.Under physiological conditions,anti-inflammatory and pro-inflammatory factors are in balance in the cells,but in the presence of surgical trauma stimuli,cyclooxygenase-2(COX-2)expression,and prostaglandin release are induced and prostaglandin receptors are activated.Prostaglandins increase the concentration of cyclic adenosine monophosphate in inflammatory cells,leading to increased expression of the pro-inflammatory factors interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α),and inhibition of the synthesis of the anti-inflammatory factor interleukin-10(IL-10),triggering an inflammatory response.Interleukin-17(IL-17)is secreted mainly by helper T cells(Th17)and is an important pro-inflammatory cytokine in the body,while IL-10 is secreted mainly by regulatory T cells(Treg)and is an important anti-inflammatory cytokine in the body,both of which together regulate the balance of the immune and inflammatory responses in the body.Previous research by the group has shown that sodium enoxaparin polymethyl methacrylate bone cement,hereafter referred to as(ES-PMMA)bone cement,as a new and improved material,does not significantly affect the biomechanical properties of the bone cement when mixed artificially with the solid powder of the bone cement in the laboratory,and can maintain the strength and stiffness of the original PMMA bone cement very well.Meanwhile,enoxaparin sodium is effectively released from the bone cement and exerts its proper drug activity.As we all know,low molecular weight heparin has been widely used in the prevention and treatment of thromboembolic diseases and in vitro anticoagulation therapy for nearly a century.However,heparin is multipotent and can be involved in the regulation of immune response and inflammatory response.The impact of heparin on the inflammatory process is one of them that is receiving an increasing amount of attention from academics,and enoxaparin sodium,as a kind of low molecular weight heparin,should have the same effect.In this study,we took advantage of the good release characteristics of ES-PMMA bone cement to explore its effect on the expression level of inflammatory factors in the rabbit knee replacement model.Objective: The effect of ES-PMMA bone cement on inflammatory factors in the animal model of knee replacement was investigated by utilizing its good release characteristics.Methods: To create a model for a knee joint replacement,40 New Zealand rabbits were selected and divided into four groups at random:traditional PMMA bone cement group,ES-PMMA bone cement group,sham operation group,and blank control group,with 10 rabbits in each group.At30,60,and 90 minutes after the corresponding bone cement was implanted,the local tissue with bone cement in the incision was taken from the traditional PMMA bone cement group and ES-PMMA bone cement group,and the same tissue was taken from the sham operation group and the blank control group.Immunohistochemical slice pathology was used to detect the expression of inflammatory cytokines IL-17 A,IL-6,IL-1β,and TNF-α in local tissues.Results: Immunohistochemical results showed that IL-17 A positive cells were expressed in the traditional PMMA bone cement group,ES-PMMA bone cement group,sham operation group,and blank control group.At 60 minutes,the expression of positive cells in the ES-PMMA bone cement group was elevated,and yet considerably less than that in the traditional PMMA bone cement group.The sham operation group displayed the greatest number of positive cells in the visual field at 90 minutes.The expression of IL-6 positive cells used to be focused in the tissue space,and the expression of IL-6 in the ES-PMMA bone cement group used to be decreased than that in the traditional PMMA group and the sham operation group at all time points.The expression of IL-1β was once the best possible in the traditional PMMA bone cement group and the sham operation group at 30 min and used to be extensively decreasing in the ES-PMMA group.There used to be no substantial distinction in the expression of TNF-α positive cells between the traditional PMMA bone cement group and the sham operation group at 30 min,and the quantity of positive cells in the ES-PMMA group used to be considerably much less than that in the traditional PMMA group and the sham operation group.Summary:1.The rabbit knee replacement model can be an established animal model that mimics the local inflammatory environment of surgery for macroscopic experiments.2.In the rabbit knee replacement model,ES-PMMA bone cement c an reduce the expression level of local tissue inflammatory factor IL-17A/IL-6/IL-1β/TNF-α by releasing enoxaparin sodium after implantation.Part Two Effects of ES-PMMA bone cement on the apoptosis of endo-thelial cells induced by lipopolysaccharide(LPS)The inflammatory response to total knee arthroplasty can lead to several complications,which are closely linked to an increase in inflammatory factors.In animal studies,we have found that the new ES-PMMA bone cement can inhibit the local inflammatory response in knee arthroplasty and modulate the local inflammatory microenvironment.In knee arthroplasty,thrombosis can be monitored in the pulmonary artery as a result of bone cement implantation,probably as a reaction to the polymerization of PMMA bone cement,resulting in a large release of heat.The stimulation of local tissues during its reaction can activate the body’s coagulation system,form microthrombi and occlude small pulmonary arteries.The results of our previous study confirmed that the activation of the coagulation system as a result of surgical manipulation leads to a localized state of hypercoagulation in the surgical area.At the same time,when the bone cement is injected into the medullary cavity and the prosthesis is inserted,the pressure increases rapidly within a short period,squeezing the microthrombi formed into the small venous circulation system and leading to serious complications such as pulmonary embolism.Endothelial cell damage is one of the hot issues discussed in the study of thrombosis and its associated diseases.Endothelial cell damage exposes the subendothelial collagen,which activates the body’s coagulation system and triggers platelet aggregation and activation,one of the necessary conditions for the development of a blood clot.Knee replacement surgery causes damage to vascular endothelial cells due to local tissue destruction and implantation of bone cement,which activates the anticoagulation system and leads to thrombosis.Since there is a close link between inflammatory factors and thrombosis,we chose endothelial cells as the object of further study to further discuss the mechanism by which ES-PMMA bone cement exerts anti-inflammatory effects to protect endothelial cells from in vitro experiments.Knee replacement surgery is very traumatic and painful,and the body has an increased inflammatory response due to stress,especially elevated levels of inflammatory factors such as IL-6 and TNF-α.A type IV hypersensitivity reaction can be triggered by the infiltration of effector T cells,macrophages,and leukocytes into the implant site and the induction of inflammation damaging the surrounding tissue.We established a model of LPS-induced endothelial cell apoptosis,and in cellular experiments,we found that ES-PMMA bone cement,as a novel material,can significantly reduce the rate of endothelial cell apoptosis through its inhibitory effect on inflammatory factors,while the reduction in endothelial cell apoptosis and necrosis reduces the opportunity for subendothelial collagen exposure,which in turn reduces platelet aggregation and activation and decreases the incidence of thrombosis,which further demonstrates that ES-PMMA bone cement has certain anti-inflammatory properties.Objective: By establishing a model of LPS-induced endothelial cell apoptosis,the mechanism by which ES-PMMA bone cement inhibits the expression of inflammatory factors and affects endothelial cell apoptosis was investigated.Methods: Bone cement was prepared as in the animal experiments,the prepared bone cement was mixed in the liquid phase and placed in1cm*1cm*1cm molds to cure for use,and the specimens were autoclaved before use.The optimal induction concentration of LPS was screened by the MTT method in pre-experiments and a cytotoxicity assay was performed;the optimal concentration of LPS was used to simulate the endothelial cell apoptosis model.The trials were classified into four groups: blank control group,LPS induction group,PMMA+LPS group,and ES-PMMA+LPS group.Endothelial cell apoptosis was investigated using flow cytometry;the levels of IL-6,TNF-α,IL-10,and intercellular adhesion molecule(ICAM)and related protein expression in cell cultures were detected by Elisa,protein blotting(Western Blot),and cellular immunofluorescence respectively.Results: When the concentration of LPS was 100mg/ml screened by the MTT method,the 24-hour cell survival rate reached 80%.With the extension of time,the cell survival rate decreased significantly,and the induction concentration was determined to be 100mg/ml in the experiment.The results of flow cytometry and Elisa were as follows: compared with the blank control group,the contents of IL-6,TNF-α,and ICAM increased(P<0.001),the content of IL-10 decreased(P<0.001),and the apoptosis rate increased(P<0.001)in LPS-induced group;the contents of IL-6,TNF-α,and ICAM in ES-PMMA+LPS group were decreased(P<0.05),the content of IL-10 was increased(P<0.05),and the apoptosis rate was decreased(P<0.001),with statistical significance.The results of Western blot and immunofluorescence showed that the expression of IL-6,TNF-α and ICAM protein in the LPs-induced group was up-regulated compared with the blank control group(P<0.001),the average fluorescence intensity was declined(P<0.001),and the expression of IL-10 protein was down-regulated(P<0.001).Average fluorescence intensity decreased(P<0.001).The expression of IL-6,TNF-α,and ICAM in the ES-PMMA+LPS group was down-regulated(P<0.05,P<0.01,P<0.001),and the expression of IL-10 was up-regulated(P<0.001)compared with that in LPS-induced and PMMA+LPS groups;the average fluorescence intensity of IL-6,TNF-α and ICAM in ES-PMMA+LPS group was lower than that in LPS-induced group(P<0.001),while the average fluorescence intensity of IL-10 was increased(P<0.01).Summary:1.In a model of LPS-induced endothelial cell apoptosis,ES-PMMA bone cement inhibited IL-6,TNF-α,and ICAM protein expression and reduced the inflammatory response.2.ES-PMMA bone cement exerts an anti-inflammatory effect by increasing the expression level of the anti-inflammatory factor IL-10 protein.3.ES-PMMA bone cement reduces endothelial cell apoptosis by a mechanism related to the expression of proteins associated with the regulation of pro-inflammatory factors.Part Three The experimental study on the polarization of macrophages induced by ES-PMMA bone cement and its anti-inflam- matory effect on the co-culture of endothelial cells and macrophagesMacrophages are widespread in the body and are often used to maintain homeostasis and fight against pathogens.They play an integral part in the body’s immune response through antigen presentation and phagocytosis and are intrinsic immune cells in the body.The polarization response of macrophages is one of the distinctive features of their involvement in the inflammatory process and underlies the role of various stimuli in the microenvironment.Macrophages can be divided into two main types,depending on surface markers and secreted cytokines: classically activated M1-type macrophages and alternatively activated M2-type macrophages.Type M1 secretes mainly pro-inflammatory factors and is involved in the inflammatory response and tissue damage;type M2 secretes mainly anti-inflammatory factors that resist the inflammatory response and carry out tissue repair.The phenotypic shift between the anti-inflammatory response and tissue repair determines the outcome of the inflammatory response.When the body is exposed to harmful stimuli,macrophages undergo M1-type polarisation with increased expression of protein markers CD16/32,CD64,CD80,CD86,and other molecules,inducing local inflammatory responses and pro-inflammatory macrophage production;M2-type is characterized by high expression of protein markers CD10,CD163,CD206 and arginase-1(Arg-1)and other molecules,which can promote tissue repair.There are multiple mechanisms of macrophage polarization,including TLR4/NF-κB,MAPK,AKT2,and STAT1 associated with the M1 type,and STAT3/6,AKT1,and PPAR-γ associated with the M2 type.Macrophages in diverse tissues are polarised in response to changes in their environment.Lipopolysaccharide(LPS)is a microbial component that prompts macrophages to polarise towards the M1 phenotype.As mentioned previously,M1-type macrophages are capable of inducing the production of pro-inflammatory-related factors such as IL-6 and tumor necrosis factor(TNF).In comparison,M2 macrophages have the capacity for anti-inflammatory responses and tissue repair.During the evolution of inflammation,macrophages are first polarised into a pro-inflammatory M1 phenotype to help the host fight pathogens.Subsequently,macrophages are polarised to form an anti-inflammatory response to the M2 phenotype and repair damaged tissue.Due to the indispensable role of macrophages in regulating the body’s immune response and tissue metabolism,disruption of macrophage polarisation may lead to many diseases.Effective analysis of the causes of macrophage polarisation disorders can be of great help in the diagnosis and treatment of the disease.Furthermore,in diseased tissues,the direction of macrophage polarisation can be altered to the desired phenotype by drug interference and regulation.Endothelial cells and macrophages are closely involved in the regulation of inflammation.Endothelial cells trigger selective growth,differentiation,and functional polarization of macrophages.In this part of the experiment,we examined the polarization trend of macrophages and established a co-culture model of endothelial cells and macrophages to analyze the apoptosis rate of endothelial cells and the expression of inflammatory factors in co-culture.ES-PMMA bone cement was found to promote macrophage M2 polarization and reduce endothelial cell apoptosis by decreasing the expression of inflammatory factors in a co-culture model with anti-inflammatory effects compared to conventional PMMA bone cement.Objective: To further investigate the anti-inflammatory mechanism of ES-PMMA bone cement by detecting the polarization trend of macrophages in cell culture fluid and the expression of inflammatory factors and apoptosis rate of endothelial cells in the co-culture model.Methods: Bone cement specimens and cell culture solution concentrations were the same as in Part II.The experiment was divided into 4 groups:blank control group,LPS induction group,PMMA+LPS group,and ES-PMMA+LPS group.Detection of macrophage polarization and endothelial cell apoptosis in co-culture systems using flow cytometry.The levels of IL-6,TNF-α,IL-10,and ICAM and the protein expression levels in the co-culture system were investigated by Elisa,protein blotting(Western Blot)and cellular immunofluorescence,respectively.Results:1.The expression level of the macrophage-associated protein CD86 was increased in the LPS-induced group compared with the blank control group(P<0.01)and decreased in the ES-PMMA+LPS group(P<0.05);CD206expression levels were down-regulated in the LPS-induced group compared with the blank control group(P<0.001),and up-regulated in the ES-PMMA+LPS group(P<0.01).2.The apoptosis rate was elevated in the LPS-induced group compared with the blank control group in the endothelial cell and macrophage co-culture system(P<0.001),and significantly decreased in the ES-PMMA+LPSinduced group compared with the LPS-induced group(P<0.001).The levels of IL-6,TNF-α,and ICAM in the supernatant were elevated in the LPS-induced group(P<0.001)and decreased in IL-10(P<0.001)in the Elisa assay compared to the blank control group.The ES-PMMA+LPS-induced group showed declined levels of the above cytokines(P<0.01 or P<0.05)and increased levels of IL-10(P<0.05)compared to the LPS-induced group.3.Western Blot and immunofluorescence results indicated that IL-6,TNF-α,and ICAM protein expression was up-regulated in the LPS-induced group compared to the blank control group(P<0.001)and the mean fluorescence intensity increased(P<0.001).IL-10 expression was downregulated(P<0.001),mean fluorescence intensity was decreased(P<0.001);IL-6,TNF-α,and ICAM protein expression were downregulated(P<0.05 or P<0.01)and IL-10 expression was upregulated in the ES-PMMA+LPS group compared with the LPS-induced group and PMMA+LPS group(P<0.001).The mean fluorescence intensity of IL-6,TNF-α,and ICAM was decreased(P<0.001)and the mean fluorescence intensity of IL-10 was increased(P<0.001)in the ES-PMMA+LPS group compared to the LPS-induced group.Summary:1.ES-PMMA bone cement can regulate the expression of macrophagespecific proteins CD86 and CD206 through the release of enoxaparin sodium,reduce M1 polarization of macrophages and promote M2 polarization,and ameliorate the inflammatory response.2.In a co-culture system of macrophages and endothelial cells,ESPMMA bone cement reduced apoptosis of endothelial cells by a mechanism associated with reduced expression of the pro-inflammatory factors IL-6 and TNF-α.3.ES-PMMA bone cement reduces the expression of the intercellular adhesion molecule ICAM protein,which inhibits the tendency of macrophages to adhere due to endothelial cell injury and inhibits further exacerbation of the inflammatory response.4.ES-PMMA bone cement increased the expression level of the antiinflammatory factor IL-10 protein in the co-culture system,further validating the experimental results in Part II.Conclusions:1.The rabbit knee replacement model can be used as a well-established animal model to mimic the local inflammatory environment of surgery.In animal experiments,modified ES-PMMA bone cement implantation reduced the secretion levels of inflammatory factors IL-17A/IL-6/IL-1β/TNF-α in the local tissues of the surgical area through the release of enoxaparin sodium.2.In the LPS-induced endothelial cell apoptosis model and macrophage and endothelial cell co-culture system,ES-PMMA bone cement inhibited the expression of pro-inflammatory factors IL-6,TNF-α,and ICAM protein,increased the expression level of anti-inflammatory factor IL-10 protein,reduced the inflammatory response and exerted anti-inflammatory effects.3.ES-PMMA bone cement reduces the inflammatory response by interfering with the expression of the characteristic macrophage proteins CD86 and CD206 and regulating the direction of their M1/M2 phenotypic polarization through the release of enoxaparin sodium.4.In in vitro experiments,both ES-PMMA bone cement reduced the rate of apoptosis of endothelial cells by a mechanism related to the regulation of protein expression related to inflammatory factors.5.ES-PMMA bone cement suppresses the tendency of macrophages to adhere due to endothelial cell injury by reducing the expression of the intercellular adhesion molecule ICAM protein,preventing a further increase in the inflammatory response.6.The modified ES-PMMA bone cement also caused a local inflammatory response,but less so than the conventional PMMA bone cement,suggesting that enoxaparin sodium has some anti-inflammatory effect. |
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