ALDH6A1 Improves Implant Osseointegration In Hyperlipidemia Mice Via The ROS/Wnt/β-catenin Pathway

Background and objectivesThe direct structural and functional connection between the ordered living bone and the surface of a load-carrying implant,which is named osseointegration,is the prerequisite for the favorable clinical performance of dental implants.Some systematic metabolic diseases are potential risk factors of implant therapy.Among them,hyperlipidemia is one of the most common systemic metabolic diseases characterized by abnormal plasma cholesterol,triglyceride,and low-density lipoprotein.But the possible impact of hyperlipidemia on titanium implant osseointegration has not attracted enough attention by clinicians.Besides,some controversies and limitations still exist in both clinical trials and basic researches.The differences existed in the current researches are attributed to various interfering factors,such as serum lipid levels,genetic defects,serum glucose,and obesity.Therefore,it is of great significance to elucidate the effects and mechanisms of hyperlipidemia on implant osseointegration by strictly controlling the experimental variables,eliminating the interfering factors,and comprehensively assessing the animal model.Reactive oxygen species(ROS)are a series of pleiotropic molecular oxygen derivatives that participate in the regulation of various biological processes.The abnormal serum lipid level induces the overproduction of ROS,leading to non-specific oxidation of intracellular proteins and various pathological changes.The Wnt/β-catenin signaling pathway is required for bone formation and has been proven to be inhibited by ROS.However,whether and how the ROS/Wnt/β-catenin pathway is involved in the hyperlipidemia-induced poor osseointegration needs further exploration.Aldehyde dehydrogenase 6 family member A1(ALDH6A1),also known as mitochondrial methylmalonate semialdehyde dehydrogenase(MMSDH),is located in the mitochondria and is mainly distributed in the liver,kidney as well as heart tissue.ALDH6A1 can bind with fatty acyl-CoA and participates in lipid metabolism.Besides,ALDH6A1 plays a key role in redox regulation by catalyzing malondialdehyde(MDA)and methylmalonic acid semialdehyde to acetyl-CoA and propionyl-CoA respectively through irreversible oxidative decarboxylation.However,whether and how ALDHA61 regulates implant osseointegration under hyperlipidemia environment needs further exploration.Therefore,the present study aimed to investigate 1)the influence of hyperlipidemia on implant osseointegration and the effects of high-fat medium on the the biological functions of mouse primary osteoblasts,2)the mechanisms underlying impaired implant osseointegration in hyperlipidemia and whether the ROS/Wnt/β-catenin pathway is involved in this process,3)the role of the key protein ALDH6A1 screened by proteomics in improving osseointegration of hyperlipidemia mice and regulating the ROS/Wnt/β-catenin pathway.Materials and methods1.The effects of hyperlipidemia on the implant osseointegration in mice and the biological functions of mouse primary osteoblastsBoth high-fat diet(HFD)and apolipoprotein E(ApoE)gene deficiency were used to establish the hyperlipidemia mice model.Body weight,body shape,serum indicators,and condition of important organs were comprehensively evaluated.Experimental implants were implanted into the distal end of bilateral femurs.After 4,8,12 weeks,the osseointegration was evaluated by Micro-CT and hard tissue section staining.The mouse primary osteoblasts were isolated,cultured,and identified.500 μM oleic acid and 500 μM palmitic acid were used to simulate the hyperlipidemia environment in vitro,and the biological functions of osteoblasts were observed.Cell proliferation ability was detected by CCK-8 and EdU fluorescent staining.Cell migration ability was detected by wound healing experiment.Cytoskeleton was observed by rhodamine phalloidin staining.Cell morphology on the titanium surface was observed by scanning electron microscope(SEM).The lipid droplets in osteoblasts were observed by Bodipy staining and Oil Red O staining.The osteogenic activity of the cells was detected by alkaline phosphatase(ALP)staining and Western blot.2.The role of the ROS/Wnt/β-catenin pathway in the hyperlipidemia-induced impaired osseointegration in mice and inhibited osteogenic activity of osteoblasts(1)Effects of the high-fat environment on the ROS level and Wnt/β-catenin pathway in osteoblasts and peri-implant tissuesIn vitro,the intracellular total ROS level(DCFH-DA staining),MDA content,mitochondrial ROS level(MitoSox staining),and mitochondrial membrane potential(MitoTracker staining)were used to detect the effects of high-fat medium on the ROS level in osteoblasts.Immunofluorescence staining and Western blot were used to detect the effect of high-fat medium on the Wnt/β-catenin pathway in osteoblasts.In vivo,immunofluorescence staining was used to detect the expression of 8-OHdG(a marker of oxidative damage)and Non-p-β-catenin(the activated form of β-catenin)around the implants in diet-induced hyperlipidemia mice.(2)The role and mechanism of N-acetyl-L-cysteine(NAC,ROS antagonist)and Wnt3a(canonical Wnt pathway activator)on improving the osseointegration and reshaping the osteblast activity in high-fat environmentIn vitro,2 mmol/L NAC or 200 ng/mL Wnt3a were added in high-fat medium for osteoblasts culture respectively.ALP staining and Western blot were used to detect the osteogenic activity of osteoblasts.Cell morphology on the titanium surface was observed by SEM.DCFH-DA staining and mitochondrial membrane potential detection(JC-1 staining)were used to detect the ROS level in osteoblasts.Immunofluorescence staining and Western blot were used to detect the effect of NAC or Wnt3a on the Wnt/β-catenin signaling pathway of osteoblasts cultured in high-fat medium.In vivo,the HFD induced hyperlipidemia mice model was established.Experimental implants were implanted into the distal end of bilateral femurs.200 μL of 10 mmol/L NAC solution or 1 μg/mL Wnt3a solution were injected into the knee joint capsule every 2 days.Four weeks after implantation,Micro-CT,hard tissue section staining,and hematoxylin and eosin(HE)staining were used to detect the osseointegration of implants.Immunofluorescence staining was used to detect the oxidation injury level,the Non-p-β-catenin expression,and the ALP expression around the implants.3.The role and mechanism of ALDH6A1 in improving the osseointegration and reshaping the osteoblast activity in high-fat environment via the ROS/Wnt/β-catenin pathway(1)Proteomic analysis of the high-fat medium impairing the biological functions of mouse primary osteoblastsThe mouse primary osteoblasts cultured in normal medium and high-fat medium were collected for proteomic detection.ALDH6A1,a potential target in the process of cell biological dysfunction caused by high-fat environment was screened by bioinformatics analysis methods.Subsequently,immunofluorescence staining,Western blot,and qRT-PCR were used to detect the changes of ALDH6A1 expression around the implants in hyperlipidemia mice and in the osteoblasts cultured in high-fat medium.(2)The effect of ALDH6A1 on the osteogenic function of osteoblasts in normal mediumSmall interfering RNA(siRNA)was used to silence ALDH6A1 in osteoblasts.Lentiviral infection technology was used to stably overexpress or knock down ALDH6A1 in osteoblasts.The changes of osteogenic activity were detected by ALP staining,Western blot,and qRT-PCR.(3)The effects and mechanisms of ALDH6A1 overexpression on improving the osseointegration and reshaping the osteoblast activity in high-fat environmentIn vitro,normal medium group(NC group),high-fat medium group(HF group),ALDH6A1 overexpression group in the high-fat medium(OE-HF group),and vehicle group in the high-fat medium(OENC-HF group)were designed.The effect of ALDH6A1 overexpression on cell proliferation in high-fat medium was detected by CCK-8 and EdU fluorescence staining.The osteogenic activity was detected by ALP staining,Western blot,and qRT-PCR.The intracellular ROS level was detected by DCFH-DA staining,MDA content detection,MitoSox staining,and Mito-Tracker staining.The activation level of the Wnt/βcatenin pathway was detected by immunofluorescence staining,Western blot,and qRT-PCR.In vivo,mice were randomly allocated to four groups:C57BL/6J normal diet group(NC group),C57BL/6J high-fat diet group(HF group),C57BL/6J high-fat diet and locally ALDH6A1 overexpression virus injection group(OE-HF group),C57BL/6J high-fat diet and locally blank vehicle injection group(OENC-HF group).Three days after implantation,ALDH6A1 overexpressing lentivirus or the blank vehicle lentivirus were injected into the knee joint capsule every 5 days.Four weeks after implantation,the osseointegration of implants were detected by Micro-CT,hard tissue section staining,and HE staining.Immunofluorescence staining was used to detect the ALDH6A1 expression,the oxidation injury level,the Non-p-βcatenin expression,and the ALP expression around the implants.Results1.Hyperlipidemia impaired the implant osseointegration in vivo and the biological functions of mouse primary osteoblasts in vitroBoth HFD feeding and ApoE gene deficiency could successfully establish the hyperlipidemia mouse model,but some differences existed between the two animal models in terms of body weight,serum lipid levels,and the health of important organs.The results of Micro-CT and hard tissue section staining showed that compared with the normal group,the trabeculae around the implants was more scattered,and the bone-implant contact ratio was significantly reduced in the hyperlipidemia group.In vitro,the mouse primary osteoblasts were successfully isolated,cultured,and identified.The high-fat medium impaired proliferation,migration and normal morphology of osteoblasts,increased intracellular lipid accumulation,and significantly inhibited the osteogenic activity of osteoblasts.2.The ROS/Wnt/β-catenin pathway played a key role in hyperlipidemia-induced poor implant osseointegrationHigh-fat medium induced ROS overproduction and inhibited the Wnt/β-catenin pathway in osteoblasts.Obvious oxidative damage and reduced Non-p-β-catenin expression were also observed around the implants in hyperlipidemia mice.In vitro,NAC or Wnt3a treatment reactivated the Wnt/β-catenin pathway and promoted the osteogenic activity of osteoblasts in high-fat medium.In vivo,NAC or Wnt3a increased the expression of Non-p-β-catenin and ALP around the implants in hyperlipidemia mice.Besides,NAC suppressed intracellular oxidative stress of osteoblasts in high-fat medium in vitro and reduced the oxidative damage level around the implants in hyperlipidemia mice in vivo,whereas Wnt3a did not.3.ALDH6A1 was a key protein that regulated the ROS/Wnt/β-catenin pathway and improved implant osseointegration in hyperlipidemic miceBioinformatics analysis revealed that ALDH6A1 was the potential target in the process of hyperlipidemia-induced poor implant osseointegration.Further in vitro and in vivo experiments showed that the expression of ALDH6A1 was significantly reduced in osteoblasts cultured in high-fat medium and around the implants in hyperlipidemia mice.Silencing and knocking down ALDH6A1 by siRNA and lentivirus respectively reduced the osteogenic activity of osteoblasts significantly,while overexpression of ALDH6A1 by lentivirus could significantly enhance the osteogenic activity of osteoblasts.The above results confirmed the important regulatory role of ALDH6A1 in the osteogenic activity of osteoblasts.In vitro,overexpression of ALDH6A1 did not affect cell proliferation in the high-fat medium,but could significantly reduce the ROS level,activate the Wnt/β-catenin pathway,and improve the osteogenic activity of osteoblasts.In vivo,overexpression of ALDH6A1 around the implants could reduce the oxidative damage,increase the expression of Non-p-β-catenin and ALP,and significantly improve the implant osseointegration in hyperlipidemia mice.Conclusions1.Both HFD-induced and genetic hyperlipidemia impaired implant osseointegration in the femur of mice.High-fat medium impaired proliferation,migration and normal morphology of osteoblasts,increased intracellular lipid accumulation,and significantly inhibited the osteogenic activity of osteoblasts.2.Hyperlipidemia impaired the implant osseointegration and the osteogenic activity of mouse primary osteoblasts via the ROS/Wnt/β-catenin pathway.3.ALDH6A1 could reshape the activity of osteoblasts and significantly improve the implant osseointegration in hyperlipidemia mice by regulating the ROS/Wnt/β-catenin pathway.