Mechanism of Youguiwan Regulating BMP-2/Smad Signaling Pathway to Promote Bone Formation in Postmenopausal Rats with Osteoporosis / 中国实验方剂学杂志

ObjectiveTo observe the effects of Youguiwan on bone metabolism and bone morphogenetic protein-2 （BMP-2）/Smad signaling pathway in ovaries-removed rats with osteoporosis and study the mechanism of Youguiwan in the prevention and treatment of osteoporosis. MethodA postmenopausal rat model of osteoporosis was prepared by bilateral ovariectomy. The 40 female SD rats were randomly divided into five groups， including sham operation group， model group， alendronate sodium group （0.1 mg·kg-1）， and high-dose and low-dose （5.36 and 2.68 g·kg-1） groups of Youguiwan. The drug was given seven days after modeling， once a day for 12 weeks. After the treatment， the changes in femur tissue structure were observed by micro-CT， including bone mineral density （BMD）， bone volume/total volume （BV/TV）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， bone surface/bone volume （BS/BV）， and trabecular separation （Tb.Sp）. Ossification was observed by saffrane-solid green staining， and serum levels of bone metabolism markers， including bone alkaline phosphatase （BALP）， osteocalcin （BGP）， type Ⅰ procollagen amino terminal propeptide （PINP）， and tartrate-resistant acid phosphatase 5b （TRACP-5b）， were determined by enzyme-linked immunosorbent assay （ELISA）. The protein and mRNA expression levels of Runx2， BMP-2， and Smad1 in rat femur were detected by Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the sham operation group， bone trabecula in the model group was sparse. BMD， BV/TV， Tb.N， and Tb.Th were decreased （P<0.05， P<0.01）. BS/BV （P<0.05） and Tb.Sp were increased. The content of BGP， BALP， PINP， and TRACP-5b in serum was significantly increased （P<0.01）. The mRNA and protein expressions of Runx2， BMP-2， and Smad1 in rat femur were significantly decreased （P<0.05， P<0.01）. Compared with the model group， the number of bone trabeculae in the high-dose and low-dose groups of Youguiwan was increased， and the bone microstructure was improved. BMD， BV/TV， Tb.N， and Tb.Th were increased significantly （P<0.05， P<0.01）， and BS/BV and Tb.Sp were increased. The content of bone metabolic markers decreased （P<0.05， P<0.01）. ConclusionYouguiwan has certain preventive and therapeutic effects on postmenopausal osteoporosis， and its mechanism may be related to promoting bone formation by regulating the BMP-2/Smad signaling pathway.

Clinical Observation on the Effect of Postmenstrual Proliferative Prescription Through Replenishing Qiand Blood on Ovulation Induction in Patients with Polycystic Ovarian Syndrome / 广州中医药大学学报

Objective To observe the effects of Postmenstrual Proliferative Prescription(mainly composed of Codonopsis Radix,Atractylodis Macrocephalae Rhizoma,Poria,Rehmanniae Radix Praeparata,Paeoniae Radix Alba,Angelicae Sinensis Radix,Chuanxiong Rhizoma,Cervi Cornu Degelatinatum,Corni Fructus,Cuscutae Semen,and Eucommiae Cortex)through replenishing qi and blood on the ovulation rate and pregnancy rate in patients with ovulatory dysfunction infertility caused by polycystic ovarian syndrome(PCOS)during ovulation-induction treatment,and to explore the therapeutic effects and possible therapeutic mechanism.Methods Sixty patients with ovulatory dysfunction infertility due to PCOS were randomly divided into a treatment group and a control group,with 30 patients in each group.The control group was given Clomifene Citrate Capsules to promote ovulation,and the treatment group was given Postmenstrual Proliferative Prescription on the basis of the ovulation-induction program of the control group starting from the fifth day of menstruation or progesterone withdrawal bleeding.The two groups were treated for one menstrual cycle as a course of treatment.The changes in the serum sex hormones of estradiol(E2),follicle-stimulating hormone(FSH),luteinizing hormone(LH),and progesterone(P)on the 2nd to 5th day of menstruation,as well as the changes in serum growth differentiation factor 9(GDF9)and bone morphogenetic protein 15(BMP15)levels on the 2nd to 5th day of menstruation and on the day of human chorionic gonadotropin(HCG)injection were observed in the two groups.Moreover,the ovulation rate,pregnancy rate and clinical efficacy of the patients in the two groups were analyzed.Results(1)No statistically significant differences in serum levels of sex hormones E2,FSH,LH and P on the 2nd to 5th day of menstruation were shown between the two groups of patients(P＞0.05).(2)On the 2nd to 5th day of menstruation and on the day of HCG injection,there were no significant differences in the serum GDF9 and BMP15 levels between the two groups(P＞0.05).(3)The ovulation rate and pregnancy rate in the treatment group were 93.33%(28/30)and 26.67%(8/30)respectively,which were significantly higher than 70.00%(21/30)and 13.33%(4/30)in the control group.And the differences tested by chi-square test were statistically significant between the two groups(P＜0.05).(4)The total effective rate of the treatment group was 93.33%(28/30),and that of the control group was 70.00%(21/30).The intergroup comparison(tested by rank sum test)showed that the therapeutic efficacy of the treatment group was significantly superior to that of the control group,and the difference was statistically significant(P＜0.05).Conclusion Postmenstrual Proliferative Prescription through replenishing qi and blood can improve the ovulation rate and pregnancy rate during ovulation-induction treatment in the patients with ovulatory dysfunction infertility due to PCOS.It is indicated that Postmenstrual Proliferative Prescription can enhance the quality of the oocytes and the potential of embryo implantation during the ovulation-induction treatment.

Adipose-derived mesenchymal stem cells overexpressing bone morphogenetic protein 2 promote alveolar bone defect repair in osteoporosis rats / 中国组织工程研究

BACKGROUND:Jaws are most vulnerable to osteoporosis.Adipose-derived mesenchymal stem cells and bone morphogenetic protein 2 have the effect of promoting bone regeneration in osteoporosis.However,the repair effect of bone morphogenetic protein 2 modified adipose-derived mesenchymal stem cells on alveolar bone defects in osteoporosis is rarely reported. OBJECTIVE:To explore the repair effect of adipose-derived mesenchymal stem cells overexpressing bone morphogenetic protein 2 on alveolar bone defects in osteoporosis rats. METHODS:(1)The rat adipose-derived mesenchymal stem cells were infected with lentivirus overexpressing bone morphogenetic protein 2 gene,and identified by detecting the expression of green fluorescent protein and bone morphogenetic protein 2.(2)Osteoporosis rat model was established by ovariectomy.A 3 mm×3 mm×3 mm cylindrical defect was prepared at the first molar position on both sides of the upper jaw.(3)Gelatin sponge was implanted in rats of the sham operation group and osteoporosis group.In the adipose-derived stem cell group,the adipose-derived mesenchymal stem cells infected with empty vector lentivirus and gelatin sponge complex were implanted.In the adipose-derived mesenchymal mesenchymal stem cell group overexpressing bone morphogenetic protein 2,a complex of adipose-derived mesenchymal stem cells overexpressing bone morphogenetic protein 2 and gelatin sponge was implanted.Relevant indexes were tested one month later. RESULTS AND CONCLUSION:(1)The transfection efficiency of the adipose-derived mesenchymal stem cell group and adipose-derived mesenchymal stem cell group overexpressing bone morphogenetic protein 2 reached more than 70%.Compared with the adipose-derived mesenchymal stem cell group,the level of bone morphogenetic protein-2 protein in the adipose-derived mesenchymal stem cell group overexpressing bone morphogenetic protein-2 was significantly higher(P<0.05).(2)A large amount of new bone could be seen in the bone defect area of the sham operation group.Compared with the sham operation group,the osteoporotic group had a small amount of new bone formation;the new bone area was significantly reduced,and alkaline phosphatase,osteocalcin,and bone morphogenetic protein 2 mRNA and protein levels were significantly reduced.Compared with the osteoporosis group,the adipose-derived mesenchymal stem cell group and the adipose-derived mesenchymal stem cell group overexpressing bone morphogenetic protein 2 had a large number of new bone formation;the area of new bone was significantly increased,and the levels of alkaline phosphatase,osteocalcin,and bone morphogenetic protein 2 mRNA and protein were significantly increased.Moreover,the adipose-derived mesenchymal stem cell group overexpressing bone morphogenetic protein 2 was superior to the adipose-derived mesenchymal stem cell group(all P<0.05).(3)The results showed that bone morphogenetic protein 2 was less expressed in the alveolar bone of osteoporosis rats,and adipose-derived mesenchymal stem cells overexpressing bone morphogenetic protein 2 could promote osteogenesis and regeneration of alveolar bone defects in osteoporosis rats.

Preparation of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold and its chondrogenic induction / 中国组织工程研究

BACKGROUND:Natural bone morphogenetic protein 2 disperses and degrades rapidly in vivo,reducing local concentration and therapeutic efficacy.Simply combining bone morphogenetic protein 2 with tissue engineering scaffolds could not stay in vivo for a long time,making it difficult to achieve good sustained and controlled release effects.OBJECTIVE:To prepare and test the biological properties and chondrogenic induction effect of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold.METHODS:SD rat tail collagen was extracted and a collagen cartilage scaffold was prepared using a vacuum freeze-drying machine chemical crosslinking method.The plasmid expressing collagen-binding domain-bone morphogenetic protein 2 was constructed by rapid cloning C112 homologous recombination,constructed by genetic engineering,and introduced into E.coli,and then collagen-binding domain-bone morphogenetic protein 2 was isolated and purified.Natural bone morphogenetic protein 2 and collagen-binding domain-bone morphogenetic protein 2 were combined with collagen cartilage scaffolds,respectively,to detect the release level of bone morphogenetic protein 2 in the scaffolds.The biocompatibility of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold was detected by CCK-8 assay and F-Actin staining.Bone marrow mesenchymal stem cells were implanted on two kinds of collagen cartilage scaffolds for chondrogenic induction,and their chondrogenic induction activity was tested.RESULTS AND CONCLUSION:(1)The binding rate of collagen-binding domain-bone morphogenetic protein 2 to collagen cartilage scaffolds was higher than that of natural bone morphogenetic protein 2(P<0.05).After being immersed in PBS for 7 days in vitro,the release of bone morphogenetic protein 2 in the collagen-binding domain bone morphogenetic protein 2-collagen cartilage scaffold was smaller than that in the natural bone morphogenetic protein 2-collagen cartilage scaffold(P<0.05).The results of the CCK-8 assay and F-Actin staining showed that the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold had no obvious cytotoxicity and had good biocompatibility.(2)After 14 days of chondrogenic induction,ELISA detection demonstrated that the expressions of agglutincan and type Ⅱ collagen A1 in the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold group were higher than those in the natural bone morphogenetic protein 2-collagen cartilage scaffold group(P<0.05).Under scanning electron microscopy,more bone marrow mesenchymal stem cells were observed on the inner wall of the pores of the two groups of scaffolds,and the cell morphology and size were the same,and the cells were closely arranged,without cell fragmentation or abnormal morphology.(3)The results indicate that the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold has good biological properties and chondrogenic induction activity.

Influence of bone microenvironment on regeneration process of tissue-engineered bone / 中国组织工程研究

BACKGROUND:Bone tissue defects are one of the most common diseases in orthopedics,and the current treatments for this disease are inadequate.The development of tissue engineering brings new hope for bone defect repair:by regulating the release of bioactive substances and the process of vascularization and neurogenesis at the defect site,it can effectively improve the microenvironment of bone tissue and promote osseointegration,which is the most promising research idea for large-size bone defect repair. OBJECTIVE:To explore the research progress of regulating bone microenvironment changes in bone defect repair in recent years from the effects of bioactive substances,vascularization and neurotization on three aspects of bone microenvironment changes,and to provide new ideas and strategies for the treatment of large-size bone defects. METHODS:The search terms"bone tissue engineering,angiogenesis,neurotization,cytokines,bone morphogenetic protein,vascular endothelial growth factor,neuropeptides,bone microenvironment"in Chinese and English were used to search for articles on the influence of changes in the bone microenvironment and their application in bone tissue engineering published from January 1,2001 to December 31,2022 on CNKI,WanFang,Web of Science,Science Direct,and PubMed.Finally,109 articles were included for review. RESULTS AND CONCLUSION:(1)The bone microenvironment is essential for the induction of bone tissue stem cell growth and differentiation,and mainly consists of the extracellular matrix of the bone tissue seeds and the biochemical factors required for intercellular interactions,the local blood circulation network and the surrounding nerve tissue.(2)Bone defect repair is a continuous process divided into multiple phases that overlap and are mediated by multiple cytokines,and the same cytokine can have mutually synergistic or antagonistic effects in one or more healing phases.(3)Neovascular regeneration is key to initiating bone repair,as neovascularisation not only provides essential nutrients,osteoblasts and growth factors for bone repair,but is also a gateway for repair cells to enter the injury zone.(4)In addition to regulating the type,dose and timeliness of vascular-inducing factor release to achieve blood transport reconstruction.The study of differential release delivery systems of multiple factors and the application of gene transfer technology will be the future research direction to solve large bone defects.(5)Neuropeptides can bind to relevant receptors and act on specific signaling pathways to guide vascular growth and influence bone healing,bone regeneration and the balance between osteogenesis and osteolysis through a variety of pathways.(6)In the establishment of neuralized tissue-engineered bone,the role of changes in the bone tissue microenvironment and neuromodulation is bidirectional.Cytokines in the bone matrix can participate in neuronal signaling pathways through the blood-nerve barrier.Neuropeptides secreted by glial cells act on the bone microenvironment,affecting bone healing,bone regeneration and the balance between osteogenesis and osteolysis.(7)There are still many questions regarding the regulation of the bone microenvironment by bioactive substances and the processes of vascularization and neurogenesis,such as the rapid diffusion and degradation of cytokines in the body and their loss of activity,the temporal and spatial distribution of angiogenesis-related growth factors,and the establishment of neurogenesis through the body's feedback regulatory mechanism,which need to be improved by subsequent studies.

Calcium phosphate combined with recombinant human bone morphogenetic protein-2 in repair and reconstruction of tibial infectious bone defects / 中国组织工程研究

BACKGROUND:Although the clinical application of Masquelet technology has achieved extensive success,the research on optimizing all aspects of Masquelet technology is still being carried out.The focus of doctors is to speed up bone healing and shorten bone healing time after bone grafting. OBJECTIVE:To observe the effect of calcium phosphate combined with recombinant human bone morphogenetic protein-2 in repairing tibial infectious bone defects. METHODS:Thirty-one patients with tibial infectious bone defects were selected from The People's Hospital of Jianyang City from June 2017 to June 2022.They were treated with the Masquelet membrane induction technique.During the second stage of operation,they were divided into a control group(n=15)and a study group(n=16)according to different bone graft materials.Patients in the control group were implanted with autologous bone/allogeneic bone particles,and those in the study group were implanted with calcium phosphate combined with recombinant human bone morphogenetic protein-2/autologous bone particles.Six months after the second stage operation,peripheral blood inflammatory indexes such as white blood cell count,C-reactive protein,and erythrocyte sedimentation rate were detected.Imaging bone healing time,bone healing X-ray score,bone defect healing classification,and adjacent joint function were recorded.The presence of nail track infection,implant absorption,pain,and infection in the bone extraction area were observed. RESULTS AND CONCLUSION:(1)White blood cell count,erythrocyte sedimentation rate,and C-reactive protein levels of the two groups 6 months after the second stage operation were significantly lower than those before the first stage operation(P<0.05).There was no significant difference in each index between the two groups(P>0.05).(2)Bone healing time in the study group was shorter than that in the control group(P<0.05).(3)The Samantha X-ray score of the study group 6 months after the second stage operation was higher than that of the control group(P<0.05).The excellent and good rate of bone defect healing and adjacent joint function of the study group was higher than that of the control group(P<0.05).There was no significant difference in the recurrence rate and complication rate between the two groups(P>0.05).(4)These findings indicate that the effect of calcium phosphate combined with recombinant human bone morphogenetic protein-2 during the second stage operation of the Masquelet membrane induction technique in the treatment of tibial infectious bone defect is good and safe.

Primary cilia/intraflagellar transport mediates mechanics-responsive signaling pathway and promotes osteogenic differentiation of bone marrow stromal stem cells / 中国组织工程研究

BACKGROUND:Mechanical stimulation has been confirmed to promote osteogenic differentiation of bone marrow stromal stem cells,but the mechanism is unknown.Primary cilia are important mechanoreceptors and regulate various signaling pathways such as TGF-β1/BMP-2/SMAD.They are likely to be important targets for mechanical regulation of bone marrow stromal stem cells. OBJECTIVE:To investigate the effect and mechanism of fluid shear stress on osteogenic differentiation of bone marrow stromal stem cells. METHODS:Rat bone marrow stromal stem cells were divided into control group,mechanical stimulation group(fluid shear mechanics intervention by shaking table),mechanical stimulation + IFT88 silencing group(mechanical stimulation + silencing IFT88 expression with siRNA).After 24 hours of intervention,qRT-PCR was utilized to determine the expression of transforming growth factor β1 and bone morphogenetic protein 2.Western blot assay was used to detect the expression of phosphorylated SMAD2/3 protein.Immunofluorescent staining of primary cilia was conducted and morphology was analyzed. RESULTS AND CONCLUSION:Shear stress stimulation could promote the transcriptional activity of transforming growth factor β1 and bone morphogenetic protein 2 genes,and increase the expression of phosphorylated SMAD2/3 protein.After siRNA interfered with primary cilia,this mechanical response effect was significantly reduced.There was a Spearman correlation between the change ratio of the primary cilium area of bone marrow stromal stem cells and the increased ratio of transforming growth factor β1 and bone morphogenetic protein 2 gene transcription.These findings indicate that primary cilia/intraflagellar transport mediates the activation of fluid shear stress-responsive transforming growth factor β1/bone morphogenetic protein 2/SMAD signaling pathway and promotes osteogenic differentiation of bone marrow stromal stem cells.

Bone morphogenetic protein-2 mediated homocysteine promotes vascular calcification / 中国组织工程研究

BACKGROUND:There is an internal relationship between hyperhomocysteinemia and vascular calcification.However,the pathogenesis of hyperhomocysteinemia promoting vascular calcification is still unclear. OBJECTIVE:To investigate the role of bone morphogenetic protein-2 in hyperhomocysteinemia-induced vascular calcification. METHODS:Human carotid wax samples were divided into a calcified group(n=29)and a non-calcified group(n=13)according to the presence or absence of calcified plaque.Sixteen ApoE-/-mice were randomly divided into a control group and a hyperhomocysteinemia group,with 8 mice in each group.Bone morphogenetic protein-2 vector was used to transfect rat thoracic artery smooth muscle A7r5 cells,and gradient concentration of homocysteine(50,100,200,and 400 μmol/L)was utilized to treat A7r5 cells.Calcification was detected by alizarin red staining and hematoxylin-eosin staining.The interaction of bone morphogenetic protein 2 with Runt-related transcription factor 2 was detected by immunofluorescence,and the expressions of bone morphogenetic protein 2,Runt-related transcription factor 2,and α-smooth muscle actin were detected by immunohistochemistry and western blot assay. RESULTS AND CONCLUSION:(1)Human carotid artery tissue staining revealed that compared with the non-calcification group,inflammatory cells increased and calcification positive rate increased in the calcification group(P<0.05).Compared with the non-calcification group,the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated,and the expression of α-smooth muscle actin was decreased in the calcification group(all P<0.05).(2)The staining of mouse arterial specimens exhibited that,the positive rate of calcified area in the hyperhomocysteinemia group was significantly higher than that in the control group(P<0.05);serum homocysteine level in the hyperhomocysteinemia group was significantly higher than that in the control group(P<0.05).Compared with the control group,the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated,and the expression of α-smooth muscle actin was decreased in the hyperhomocysteinemia group(all P<0.05).(3)A7r5 cell culture analysis demonstrated that with the increase of homocysteine concentration gradient,the degree of calcification,the content of bone morphogenetic protein-2 and Runt-related transcription factor 2 protein in A7r5 cells increased(P<0.05),and the content of α-smooth muscle actin protein decreased(P<0.05).(4)The A7r5 cell culture analysis of overexpressed bone morphogenetic protein 2 showed that the calcification degree of the overexpressed bone morphogenetic protein 2 group was increased compared with the corresponding control group,the β-sodium glycerophosphate group,and the homocysteine group.RUNt-related transcription factor 2 expression up-regulated(P<0.05)and α-smooth muscle actin expression down-regulated(P<0.05).(5)The expression of bone morphogenetic protein 2 increased in A7r5 cells cultured with homocysteine in calcified medium,and the expression of Runt-related transcription factor 2 increased with the increase of bone morphogenetic protein 2 expression.(6)The results confirm that bone morphogenetic protein-2 is a key target gene in the regulation of smooth muscle cell phenotypic transformation resulting in vascular calcification by hyperhomocysteinemia.Targeted regulation of bone morphogenetic protein-2 reduces hyperhomocysteinemia-induced vascular calcification.

Cell-of-origin for heterotopic ossification induced by bone morphogenetic protein 4 in skeletal muscle / 中国组织工程研究

BACKGROUND:Heterotopic ossification of skeletal muscle is a clinically serious complication.For heterotopic ossification of skeletal muscles,the cells involved in the process of heterotopic ossification remain unclear. OBJECTIVE:To investigate the involvement of myocytes,fascia cells,and endothelial cells in the process of heterotopic ossification in skeletal muscle and to observe the cell origin of heterotopic ossification in skeletal muscle induced by bone morphogenetic protein 4. METHODS:Both C2C12 cells and the myotubes formed by the C2C12 cells in the induction medium were cultured,and 500 ng/mL bone morphogenetic protein 4 was added to the medium respectively,and whether the C2C12 cells and myotubes continued to proliferate within 10 days under the treatment were observed under a microscope.Myogenic cells(L6,derived from rats)and fibroblast-derived cells(derived from human)were co-cultured.After treatment with 500 ng/mL bone morphogenetic protein 4 and 10 ng/mL transforming growth factor-β,osteogenic and chondrogenic differentiation potential within 21 days were observed using Safranine O staining and Alcian blue staining.Using transgenic animal FVB/N-TgN(TIE2-LacZ)182Sato mice,15 μL of adeno-associated virus-bone morphogenetic protein 4(5×1010 PFU/mL)were implanted in the thigh muscle space of genetic mice for 10 and 14 days.X-gal staining was used to observe the formation of new blood vessel endothelium in the differentiated bone. RESULTS AND CONCLUSION:(1)Bone morphogenetic protein 4 caused myotube breakdown and increased C2C12 cell proliferation.Compared with other groups,the pure fibroblast-derived cell group had a higher area of positive alcian blue and safarin O staining(P<0.05)and a lower area of alkaline phosphatase staining(P<0.05),while the pure L6 group had a bigger area of alkaline phosphatase staining(P<0.05)but a smaller area of positive alcian blue and safarin O staining(P<0.05).(2)Transplantation of adeno-associated virus-bone morphogenetic protein 4-adsorbed gelatin sponge into FVB/N-TgN(TIE2-LacZ)182Sato mice resulted in heterotopic ossification.(3)X-gal staining results demonstrated that there was no obvious staining in chondrocytes and differentiated bones and Tie2+ endothelial cells did not participate in the formation of the alienated bone.(4)These findings verify that fibroblasts are the primary source of osteoblasts during the adeno-associated virus-bone morphogenetic protein 4-induced ectopic endochondral ossification in skeletal muscle,but myogenic cells are the main source of osteoblasts.Tie2+ endothelial cells might not be the cell source for cartilage and bone.

Molecular biological mechanism of acquired heterotopic ossification / 中国组织工程研究

BACKGROUND:Heterotopic ossification is a dynamic growth process.Diverse heterotopic ossification subtypes have diverse etiologies or induction factors,but they exhibit a similar clinical process in the intermediate and later phases of the disease.Acquired heterotopic ossification produced by trauma and other circumstances has a high incidence. OBJECTIVE:To summarize the molecular biological mechanisms linked to the occurrence and progression of acquired heterotopic ossification in recent years. METHODS:The keywords"molecular biology,heterotopic ossification,mechanisms"were searched in CNKI,Wanfang,PubMed,Embase,Web of Science,and Google Scholar databases for articles published from January 2016 to August 2022.Supplementary searches were conducted based on the obtained articles.After the collected literature was screened,131 articles were finally included and summarized. RESULTS AND CONCLUSION:(1)The occurrence and development of acquired heterotopic ossification is a dynamic process with certain concealment,making diagnosis and treatment of the disease difficult.(2)By reviewing relevant literature,it was found that acquired heterotopic ossification involves signaling pathways such as bone morphogenetic protein,transforming growth factor-β,Hedgehog,Wnt,and mTOR,as well as core factors such as Runx-2,vascular endothelial growth factor,hypoxia-inducing factor,fibroblast growth factor,and Sox9.The core mechanism may be the interaction between different signaling pathways,affecting the body's osteoblast precursor cells,osteoblast microenvironment,and related cytokines,thereby affecting the body's bone metabolism and leading to the occurrence of acquired heterotopic ossification.(3)In the future,it is possible to take the heterotopic ossification-related single-cell osteogenic homeostasis as the research direction,take the osteoblast precursor cells-osteogenic microenvironment-signaling pathways and cytokines as the research elements,explore the characteristics of each element under different temporal and spatial conditions,compare the similarities and differences of the osteogenic homeostasis of different types and individuals,observe the regulatory mechanism of the molecular signaling network of heterotopic ossification from a holistic perspective.It is beneficial to the exploration of new methods for the future clinical prevention and treatment of heterotopic ossification.(4)Meanwhile,the treatment methods represented by traditional Chinese medicine and targeted therapy have become research hotspots in recent years.How to link traditional Chinese medicine with the osteogenic homeostasis in the body and combine it with targeted therapy is also one of the future research directions.(5)At present,the research on acquired heterotopic ossification is still limited to basic experimental research and the clinical prevention and treatment methods still have defects such as uncertain efficacy and obvious side effects.The safety and effectiveness of relevant targeted prevention and treatment drugs in clinical application still need to be verified.Future research should focus on clinical prevention and treatment based on basic experimental research combined with the mechanism of occurrence and development.

Atf7ip is a negative regulator of bone morphogenetic protein 2 promoting osteogenic differentiation in mouse embryonic adult cells / 中国组织工程研究

BACKGROUND:Whether activating transcription factor 7 interacting protein(Atf7ip)is involved in the regulation in osteogenic differentiation is still controversial,and studying its impact on osteogenic differentiation and its specific mechanisms is of great significance. OBJECTIVE:To investigate the effect of Atf7ip on bone morphogenetic protein 2 promoting osteogenic differentiation of mouse embryonic osteoblast precursor cells(MC3T3-E1). METHODS:MC3T3-E1 cells cultured in vitro were divided into three groups:normal group,interference group(NC-siRNA group,Atf7ip-siRNA group),and high expression group(CMV-VC group and CMV-Atf7ip group),and were transfected for 24 hours,and then treated with 200 ng/mL bone morphogenetic protein 2 for 0,12,24,and 48 hours,respectively.qRT-PCR was used to detect the mRNA expression levels of Atf7ip,alkaline phosphatase,osteocalcin,type I collagen α1 in the cells of each group.Western blot assay was used to detect the protein expression of osteogenic differentiation markers Sp7 and Runx2,and the expression of Atf7ip binding molecule SETDB1,histone H3 and H3K9me3.Alkaline phosphatase activity was detected by alkaline phosphatase staining. RESULTS AND CONCLUSION:(1)With the increase of bone morphogenetic protein 2 treatment time,the protein and mRNA expression of Atf7ip decreased,while the protein expression of Sp7,Runx2 and the mRNA expression of osteocalcin and alkaline phosphatase increased significantly(P<0.05).There was no significant change in the protein expression of Atf7ip binding molecule SETDB1.(2)Compared with the NC-siRNA group,the protein expression of Sp7,Runx2 and the mRNA expression of osteocalcin and type I collagen α1 were significantly up-regulated(P<0.05),and alkaline phosphatase activity was significantly enhanced;and H3K9 methylation significantly decreased in the Atf7ip-siRNA group(P<0.05).(3)Compared with the CMV-VC group,the protein expression of Sp7 and Runx,as well as mRNA expression of osteocalcin,alkaline phosphatase,and type I collagen α1 was significantly downregulated(P<0.05),and the alkaline phosphatase activity was significantly reduced in the CMV-Atf7ip group,while the H3K9 methylation protein in the CMV-Atf7ip group was significantly upregulated compared to the control group(P<0.05).(4)In conclusion,Atf7ip expression was decreased during bone morphogenetic protein 2-induced osteogenic differentiation of MC3T3-E1,and osteogenic differentiation was significantly increased after knockdown of Atf7ip.Overexpression of Atf7ip significantly weakened osteogenic differentiation,indicating that Atf7ip is a negative regulatory factor of bone morphogenetic protein 2 promoting osteogenic differentiation of MC3T3-E1 cells.

Overexpression of long non-coding RNA Gm16104 affects osteogenic differentiation of C3H10T1/2 mesenchymal stem cells / 中国组织工程研究

BACKGROUND:The osteogenic differentiation of mesenchymal stem cells is regulated by a variety of molecules.Long non-coding RNA(lncRNA)has attracted much attention because they can participate in regulating a variety of biological processes,but the regulatory role of lncRNA on osteogenic differentiation of mesenchymal stem cells has not been fully explored. OBJECTIVE:To explore the effect of lncRNA Gm16104 on osteogenic differentiation of C3H10T1/2 mesenchymal stem cells. METHODS:The C3H10T1/2 mesenchymal stem cells were induced into osteogenic differentiation by bone morphogenetic protein-2.Alkaline phosphatase staining was performed to identify the osteogenic differentiation of the cells 5 days after osteogenic induction.Expression levels of alkaline phosphatase and lncRNA Gm16104 were detected by qRT-PCR after 0,1,3,and 5 days of osteogenic differentiation.After transfection of the overexpression plasmid of pcDNA-Gm16104,the osteogenic differentiation was identified by alkaline phosphatase staining and qRT-PCR 4 days after osteogenic induction.The expression levels of osteogenesis-related signalling pathway proteins were detected by western blot assay. RESULTS AND CONCLUSION:(1)After 5 days of osteogenic induction,alkaline phosphatase activity was significantly increased.(2)Compared with 0 days,expression levels of the osteogenic marker gene alkaline phosphatase increased and expression levels of Gm16104 decreased after 1,3,and 5 days of osteogenic induction.(3)Transfection of C3H10T1/2 cells with pcDNA-Gm16104 plasmid significantly increased the expression level of Gm16104.(4)Overexpression of Gm16104 inhibited alkaline phosphatase activity,the expression levels of the osteogenic marker gene alkaline phosphatase and the osteogenesis-related transcription factor Osterix.(5)Overexpression of Gm16104 inhibited phosphorylated protein expression of PI3K and Akt.(6)The above results suggest that overexpression of Gm16104 may inhibit osteogenic differentiation of C3H10T1/2 mesenchymal stem cells through the PI3K/Akt signaling pathway.

Vascular endothelial growth factor 165/bone morphogenetic protein improves osteoblast injury under hypoxic and reoxygenated conditions / 中国组织工程研究

BACKGROUND:It has been found that vascular endothelial growth factor 165 and bone morphogenetic proteins interact with each other during hypoxia-reoxygenation and are involved in the repair process of osteoblast injury by regulating the activation of intracellular signaling pathways. OBJECTIVE:To further investigate the relationship between vascular endothelial growth factor 165/bone morphogenetic protein and hypoxic-reoxygenated osteoblast injury. METHODS:Osteoblasts were selected and the hypoxic-reoxygenated injury model was established.Vascular endothelial growth factor 165 and bone morphogenetic protein expressions at mRNA and protein levels were detected by real-time PCR and western blot before and after modeling.After modeling,osteoblasts were given different concentrations of vascular endothelial growth factor 165 and bone morphogenetic protein 2(10,20,40 ng/mL).Cell proliferation was detected by cell counting kit-8 method and apoptosis was detected by DAPI at 12,24,36,48,and 72 hours after treatment. RESULTS AND CONCLUSION:Compared with before modeling,the mRNA and protein expressions of vascular endothelial growth factor 165 and bone morphogenetic protein 2 in osteoblasts after modeling were significantly decreased(P＜0.05).The proliferation rate of osteoblasts was significantly increased with the increase of vascular endothelial growth factor 165 concentration(P＜0.05),while the apoptosis rate of osteoblasts decreased significantly with the increase of vascular endothelial growth factor 165 concentration(P＜0.05).The proliferation rate of osteoblast was significantly increased with the increase of bone morphogenetic protein 2 concentration(P＜0.05),while the apoptosis rate of osteoblast decreased significantly with the increase of bone morphogenetic protein 2 concentration(P＜0.05).To conclude,vascular endothelial growth factor 165 and bone morphogenetic protein are lowly expressed in hypoxic-reoxygenated osteoblast injury,and treatment with vascular endothelial growth factor 165 and bone morphogenetic protein can reduce the injury of hypoxic-reoxygenated osteoblast in a concentration-dependent manner,suggesting that vascular endothelial growth factor 165 and bone morphogenetic protein have a significant protective effect against the injury of hypoxic-reoxygenated osteoblasts.

Application value of bone cement containing rhbFGF and RHBMP-2 in PKP treatment of osteoporotic lumbar compression fracture / 中国骨伤

OBJECTIVE@#To investigate the effect of bone cement containing recombinant human basic fibroblast growth factor (rhbFGF) and recombinant human bone morphogenetic protein-2 (rhBMP-2) in percutaneous kyphoplasty(PKP)treatment of osteoporotic vertebral compression fracture(OVCF).@*METHODS@#A total of 103 OVCF patients who underwent PKP from January 2018 to January 2021 were retrospectively analyzed, including 40 males and 63 females, aged from 61 to 78 years old with an average of (65.72±3.29) years old. The injury mechanism included slipping 33 patients, falling 42 patients, and lifting injury 28 patients. The patients were divided into three groups according to the filling of bone cement. Calcium phosphate consisted of 34 patients, aged(65.1±3.3) years old, 14 males and 20 females, who were filled with calcium phosphate bone cement. rhBMP-2 consisted of 34 patients, aged (64.8±3.2) years old, 12 males and 22 females, who were filled with bone cement containing rhBMP-2. And rhbFGF+rhBMP-2 consisted of 35 patients, aged (65.1±3.6) years old, 14 males and 21 females, who were filled with bone cement containing rhbFGF and rhBMP-2. Oswestry disability index (ODI), bone mineral density, anterior edge loss height, anterior edge compression rate of injured vertebra, visual analog scale (VAS) of pain, and the incidence of refracture were compared between groups.@*RESULTS@#All patients were followed for 12 months. Postoperative ODI and VAS score of the three groups decreased (P<0.001), while bone mineral density increased (P<0.001), anterior edge loss height, anterior edge compression rate of injured vertebra decreased first and then slowly increased (P<0.001). ODI and VAS of group calcium phosphate after 1 months, 6 months, 12 months were lower than that of rhBMP-2 and group rhbFGF+rhBMP-2(P<0.05), bone mineral density after 6 months, 12 months was higher than that of rhBMP-2 and group calcium phosphate(P<0.05), and anterior edge loss height, anterior edge compression rate of injured vertebra of group rhbFGF+rhBMP-2 after 6 months and 12 months were lower than that of group rhBMP-2 and group calcium phosphate(P<0.05). There was no statistical difference in the incidence of re-fracture among the three groups (P>0.05).@*CONCLUSION@#Bone cement containing rhbFGF and rhBMP-2 could more effectively increase bone mineral density in patients with OVCF, obtain satisfactory clinical and radiological effects after operation, and significantly improve clinical symptoms.

KDM4A promotes the migration and invasion of breast cancer cell line MDA-MB-231 by downregulating BMP9 / 中国癌症杂志

Background and purpose:Exogenous bone morphogenetic protein 9(BMP9)inhibits the malignant progression of human breast cancer,but its expression is often abnormally low in breast cancer.In this study,we intended to explore the expression and role of epigenetically-modified histone lysine-specific demethylase 4A(KDM4A)in breast cancer,and to investigate the relationship between KDM4A and BMP9 and its possible regulatory mechanism.Methods:The expression of KDM4A in breast cancer and its relationship with BMP9 were analyzed by bioinformatics and verified by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot.Chromatin immunoprecipitation(ChIP)verified the regulatory role of KDM4A on BMP9,and RNA stability experiments and CHX protein stability experiments verified the effect of KDM4A in BMP9 expression.Exogenous recombinant MDA-MB-231 cells transfected with KDM4A small interfering RNA(siKDM4A)or infected with siBMP9 adenovirus(Ad-siBMP9)were constructed using RNA interference technology and adenoviruses knocking down BMP9,and the migratory and invasive abilities of the cells were detected by scratch healing assay and transwell assay,respectively.Results:Bioinformatics analysis showed that the expression of KDM4A was significantly higher in breast cancer than in normal tissues,and there was a negative correlation between the expression of KDM4A and that of BMP9 in breast cancer;RTFQ-PCR and Western blot showed that KDM4A was highly expressed in different breast cancer cell lines,and the knockdown of KDM4A significantly up-regulated BMP9.ChIP experiment confirmed that KDM4A could be significantly enriched in the promoter region of BMP9 gene,reducing its histone lysine 36 position instead of position 4 methyl status,thus silencing the expression of BMP9.RNA stability assay and CHX protein stability assay confirmed that KDM4A had no significant effect on the mRNA of BMP9,but could affect its protein degradation.After knocking down KDM4A,the migration and invasion abilities of breast cancer cells MDA-MB-231 were significantly inhibited,and this effect could be partially reversed by knocking down BMP9.Conclusion:KDM4A is highly expressed in breast cancer and breast cancer cell MDA-MB-231,and can silence its expression by down-regulating the level of histone methylation in the promoter region of the BMP9 gene,as well as affecting the stability of BMP9 at the protein level rather than at the level of mRNA,and promoting the migration and invasion of breast cancer.

Bone morphogenetic protein 7 attenuates renal fibrosis in diabetic kid-ney disease rats by down-regulating Ajuba / 中国病理生理杂志

AIM:Bone morphogenetic protein 7(BMP7)reduces the expression of Yes-related protein 1(YAP1)by down-regulating Ajuba level and decreasing extracellular matrix(ECM)deposition.This study aimed to inves-tigate the influence of these factors on modifying the degree of renal fibrosis in rats with diabetic nephropathy.METH-ODS:Eighteen Sprague-Dawley(SD)rats were randomly divided into three groups:the normal control(NC)group,the diabetes mellitus(DM)group,and the DM group treated with BMP7 overexpressing adeno-associated virus(DM+rAAV-BMP7).Each group consisted of six rats.Diabetic kidney disease(DKD)was established in the DM and DM+rAAV-BMP7 groups by injecting 55 mg/kg streptozotocin(STZ)via the tail vein.NRK-52E cells were divided into three groups:the normal glucose(NG)group,the high glucose(HG)group,and the high glucose group treated with recombinant hu-man BMP7(HG+rhBMP7)group.Pathological changes in renal tissues were observed using hematoxylin and eosin(HE)and Sirius red staining.Immunohistochemical staining was performed to examine the expression sites of Ajuba and YAP1 in the renal cortex.Western blot analysis was conducted to determine the expression levels of BMP7,Ajuba,YAP1,colla-gen type Ⅲ(Col-Ⅲ),and fibronectin(FN)in the rat renal cortex and NRK-52E cells.RT-qPCR was used to measure the mRNA levels of Ajuba and YAP1 in the rat renal cortex.RESULTS:Biochemical indices revealed significantly ele-vated levels of blood glucose,serum creatinine,triglycerides,total cholesterol,and 24-hour urinary protein in the DM group compared to the NC group(P<0.05).In the DM+rAAV-BMP7 group,the levels of serum creatinine,24-hour uri-nary protein,triglycerides,and total cholesterol were lower than those in the DM group(P<0.05).Pathological staining demonstrated that the renal interstitium of the DM group exhibited inflammatory cell infiltration,fibrous tissue,collagen fi-ber deposition,disordered renal tubule arrangement,atrophy,and vacuolar degeneration,which were ameliorated in the DM+rAAV-BMP7 group.Immunohistochemistry revealed that Ajuba and YAP1 were mainly expressed in the cytoplasm and nucleus,with high expression in the cytoplasm of the DM group,which was significantly decreased in the DM+rAAV-BMP7 group.Western blot results indicated that the protein levels of FN,Col-Ⅲ,Ajuba,and YAP1 were up-regulated in the DM and the HG groups(P<0.05),but significantly down-regulated in the DM+rAAV-BMP7 group(P<0.05).RT-qP-CR results demonstrated that the mRNA levels of Ajuba and YAP1 were higher in the DM group and significantly lower in the DM+rAAV-BMP7 group(P<0.05).CONCLUSION:The overexpression of BMP7 can ameliorate renal fibrosis in rats with DKD.This effect is likely mediated by the down-regulation of Ajuba,reduction of YAP1 expression,and subse-quent inhibition of ECM deposition.

Role of AKE/GB30 cage loaded with naringin in the repair of spinal bone defect model and its effect on BMPs VEGF signaling pathway / 中国脊柱脊髓杂志

Objectives:To observe the repair effect of AKE/GB30 mesh cage loaded with naringin in spinal bone defect model,and explore the mechanism of this biomaterial based on bone morphogenetic proteins(BMPs)-vascular endothelial growth factor(VEGF)signal pathway.Methods:A dental drill was used to make a 7mmx5mmx4mm spinal bone defect model between L5 and L6 vertebrae in 30 New Zealand male rabbits,and AKE/GB30 mesh cages were prepared.The biomechanical properties of AKE/GB30 mesh cages loaded with naringin were tested,and their in vitro drug release behavior was measured.New Zealand rabbits that were successfully modeled were randomly divided into three groups using a randomly digital table method,namely,blank group,autologous bone graft group,and bone graft biomaterial+naringin combined group.Except the blank group,autologous bone transplantation and AKE/GB30 mesh cage loaded with naringin were used for repair.At 6 weeks and 12 weeks after surgery,5 rabbits were taken from each group,and the bone repair status[including bone volume/tissue volume(BV/TV),bone trabecular thickness(Tb.Th)and bone trabecular number(Tb.N)]were detected by micro computed tomography(Micro CD.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect the expressions of bone morphogenetic protein 2(BMP2),VEGF,Runt related transcription factor 2(RUNX2),alkaline phosphatase(ALP),and osteocalcin(OCN)messenger ribonucleic acid(mRNA).Immunoblotting assay(WB)was used to detect the expressions of BMP2,VEGF,RUNX2,ALP,and OCN proteins in bone tissues.Results:AKE/GB30 mesh cages had been successfully manufactured,and its characteristic testing results met the requirements for repairing spinal bone defects.The AKE/GB30 mesh cage loaded with naringin had a maximum compressive strength of 28MPa and a maximum resistance pressure of 15N.At 6 weeks,its cumulative release rate reached(98.15±1.47)％.After 12 weeks,the BV/TV,Tb.Th,and Tb.N,as well as the mRNA and protein expressions of BMP2,VEGF,RUNX2,ALP,and OCN in bone tissues of each group were higher than those after 6 weeks(P＜0.05).The above indicators in the autologous bone graft group and the bone graft biomaterial+naringin combined group were higher than those in the blank group(P＜0.05),and there were no significant differences in the above indicators between the autologous bone graft group and the bone graft biomaterial+naringin combined group(P＞0.05).Conclusions:The effect of AKE/GB30 cage loaded with naringin in repairing spinal bone defect models is equivalent to that of autologous bone graft,which is presumed to achieve by promoting the expressions of BMP2,VEGF,RUNX2,ALP,and OCN.

Osteoprotective Effect and Mechanisms of Taxillus chinensis from Different Hosts on Ovariectomized Osteoporotic Rats / 中国实验方剂学杂志

ObjectiveTo compare the effects of Taxillus chinensis from different hosts with different meridian affinity on bone microstructure and bone metabolism in ovariectomized osteoporotic rats， and investigate its mechanism of action. MethodEighty-eight specific-pathogen-free （SPF）-grade female Sprague-Dawley （SD） rats were selected and randomly divided into 11 groups： sham-operated group， model group， low-， medium- and high-dose groups of T. chinensis from Morus alba （2.5， 5， and 10 g·kg-1）， low-， medium- and high-dose groups of T. chinensis from Cinnamomum cassia （2.5， 5， and 10 g·kg-1）， and low-， medium- and high-dose groups of T. chinensis from C. burmannii （2.5， 5， and 10 g·kg-1）. After 12 weeks of drug intervention， the rats were examined for proximal femur bone density and bone microstructure using dual-energy X-ray absorptiometry （DXA） and micro-computed tomography （Micro-CT）. Histopathological changes in rat femur were observed by the hematoxylin-eosin staining （HE）. Contents of serum estradiol （E2）， bone Gla protein （BGP）， bone alkaline phosphatase （BALP）， tartrate-resistant acid phosphatase 5b （TRACP-5b） and pre-collagen type Ⅰ amino-terminal protopeptide （PINP） were measured by the enzyme-linked immunosorbent assay （ELISA）. Real-time quantitative polymerase chain reaction （Real-time PCR） was employed to detect the messenger ribonucleic acid （mRNA） expressions of bone morphogenetic protein-2 （BMP-2）， Smad1， Smad9 and recombinant runt-related transcription factor 2 （Runx2） in rat humerus. Western blot was used to detect the protein expressions of BMP-2， p-Smad1/5/9 and Runx2 in rat humerus. ResultCompared with that in the sham-operated group， the femur microstructure of rats in the model group was significantly disrupted， with significant decreases in bone mineral density （BMD） value， bone volume fraction （BV/TV）， trabecular number （Tb.N）， and trabecular thickness （Tb.Th） （P<0.01）， and significant increases in trabecular separation （Tb.Sp） and structure model index （SMI） （P<0.01）. The serum levels of BGP， BALP， TRACP-5b and PINP were significantly increased （P<0.05， P<0.01）， and E2 levels were significantly decreased （P<0.01）. The mRNA expressions of BMP-2， Smad1， Smad9， and Runx2 were significantly decreased in rat humerus （P<0.01）， and the protein expressions of BMP-2， p-Smad1/5/9， and Runx2 were significantly reduced （P<0.01）. Compared with the model group， the administration groups of T. chinensis from different hosts all elevated the BMD， BV/TV， Tb.N， Tb.Th， Tb.Sp， and SMI levels in the femur， improved bone microstructure， increased serum E2 levels （P<0.05， P<0.01）， lowered the levels of serum BGP， BALP， TRACP-5b， and PINP， upregulated the mRNA expression of BMP-2， Smad1， and Runx2 and upregulated the mRNA expression levels of Smad9 （P<0.05， P<0.01）， and upregulated the protein expressions levels of BMP-2， p-Smad1/5/9， and Runx2 （P<0.01）. The best effect was observed in the group of T. chinensis from C. cassia. ConclusionT. chinensis from different hosts improved osteoporosis in ovariectomized rats， with the group of T. chinensis from C. cassia being the most potent among the administered groups， and its treatment of osteoporosis may regulate the balance of bone conversion by regulating BMP/Smad signaling pathway.

Berberine derivative WJCPR11 enhances osteoblast differentiation

Berberine is a phytocompound from plants viz. Phellodendri cortex and Coptis rhizome, used to treat a variety of diseases. It is effective in preventing osteoporosis, but it is less effective than drugs currently used in clinical practice. In this study, we used a novel berberine derivative, WJCPR11, to promote osteoblast differentiation and to investigate its use in the prevention and treatment of osteoporosis. WJCPR11 at a safe concentration without toxicity increased alkaline phosphatase (ALP) activity induced by bone morphogenetic protein 2 (BMP2) dose-dependently. The mRNA expression of ALP, osteocalcin (OC), runt-related transcription factor 2 (Runx2), and osterix was increased, with the ALP level increasing the most. In addition, the protein abundance of bone sialoprotein (BSP), collagen, type I, alpha 1, Runx2, and osterix were also increased. Moreover, the transcriptional activity of ALP, BSP, and OC was increased by WJCPR11, with OC showing the most significant increase. The results indicate that osteoblast differentiation is promoted by WJCPR11, and it could play a role in the prevention of osteoporosis.

Micro/nano hierarchical structure of titanium surface promotes MC3T3-E1 adhesion proliferation and drug loading potential evaluation / 口腔疾病防治

Objective @# To investigate the effect of micro/nano hierarchical structures on the adhesion and proliferation of MC3T3-E1 cells, evaluate the drug delivery potential of titanium surfaces, and provide a reference for the modification of selected areas of titanium surfaces to enhance drug delivery and slow drug release. @*Methods @# Pure titanium samples (10 mm in diameter and 2.5 mm in thickness) were randomly divided into a polished group (T), anodized group (TO), and micro/nano hierarchical structure group (FTO) according to the surface treatment of the titanium. The T group was polished, the TO group was treated with anodic oxidation technology, and the FTO group was treated by femtosecond laser etching combined with anodic oxidation technology. The three surface morphologies were observed by scanning electron microscopy (SEM), the wettability of the surface was measured by the contact angle, and the surface chemical composition was analyzed by X-ray energy dispersive spectroscopy (EDS). The depth of the FTO structure and the surface roughness were measured by confocal laser scanning microscopy (CLSM). MC3T3-E1 cell adhesion proliferation and differentiation on the surface of each group of samples was assessed by immunofluorescence staining, CCK-8, and semiquantitative analysis of Alizarin staining. A freeze-drying method was applied to load recombinant human bone morphogenetic protein-2 (rhBMP-2), and an enzyme-linked immunosorbent assay (ELISA) was used to assess the drug-loading potential of different surface structures. @* Results@#SEM revealed that the surface of T group titanium plates showed uniform polishing marks in the same direction. The surface of the TO group was a nanoscale honeycomb-like titanium dioxide (TiO2) nanotube structure, and the FTO group formed a regular and ordered micro/nano layered structure. The contact angle of the FTO group was the smallest at 32° ± 1.7°. Its wettability was the best. The average depth of the first-level structure circular pores was 93.6 μm, and the roughness was 1.5-2 μm. The TO and FTO groups contained a high percentage of oxygen, suggesting TiO2 nanotube formation. The FTO group had the most significant surface cell proliferation (P<0.001) and the largest cell adhesion surface area (P<0.05). rhBMP-2 was slowly released for 14 d after loading in the FTO group and promoted extracellular matrix mineralization (P<0.001). @*Conclusion @#Titanium surface microprepared hierarchical structure has the effect of promoting MC3T3-E1 cell adhesion, proliferation, and osteogenic differentiation with drug loading potential, which is a new method of titanium surface treatment.