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.

Irisin alleviates palmitic acid-induced osteogenic inhibition in bone marrow mesenchymal stem cells / 中国组织工程研究

BACKGROUND:Irisin,a myokine isolated from the transmembrane protein FNDC5 by muscle cells during exercise,has the function of inducing the browning of white adipose tissue,but its effect on lipotoxicity-induced osteogenic differentiation and the mechanism is unclear. OBJECTIVE:To investigate the effect of irisin on the osteogenic ability of palmitic acid-induced bone marrow mesenchymal stem cells and the mechanism of action. METHODS:CCK-8 assay was used to detect the effect of different concentrations of palmitic acid on the proliferation of mouse bone marrow mesenchymal stem cells and the effect of irisin on the proliferation of mouse bone marrow mesenchymal stem cells in the presence of palmitic acid.After pretreatment with irisin and palmitic acid for 24 hours,osteogenic differentiation of mouse bone marrow mesenchymal stem cells was induced by alkaline phosphatase staining as well as qRT-PCR was performed to detect the expression of osteogenesis-related genes on day 7 of osteogenic induction culture.The expression of proteins related to the AMPK/BMP2/SMAD signaling pathway was detected by western blot assay.Alizarin red staining was conducted on day 21 to detect osteogenic differences. RESULTS AND CONCLUSION:(1)The CCK-8 assay results suggested that the amplification of bone marrow mesenchymal stem cells was inversely proportional to the concentration of palmitic acid,but at 0.02 mmol/L concentration,palmitic acid had no significant effect on the amplification of bone marrow mesenchymal stem cells,and irisin did not affect the proliferation of bone marrow mesenchymal stem cells when its mass concentration was in the range of 0.1-20 μg/L.(2)Alkaline phosphatase staining and alizarin red staining showed that palmitic acid inhibited the osteogenic differentiation ability of bone marrow mesenchymal stem cells.Irisin improved palmitic acid-induced osteogenic inhibition of bone marrow mesenchymal stem cells.qRT-PCR results showed that palmitic acid could cause the downregulation of osteogenic-related genes,and irisin could inhibit this trend.(3)Western blot assay results showed that compared with the palmitic acid intervention group,irisin treatment enhanced AMPK/BMP2/SMAD signal transduction in bone marrow mesenchymal stem cells.It is found that irisin can improve the osteogenic differentiation ability of bone marrow mesenchymal stem cells pretreated with palmitic acid,and proposed that the specific mechanism might be mediated by AMPK/BMP/SMAD signaling pathway.

Isopsoralen Promotes Osteoblast Proliferation and Differentiation via BMP2/Runx2/Osx Signaling Pathway / 世界科学技术-中医药现代化

Objective The purpose of this study was to examine effects of Isopsoralen on the osteoblast proliferation and differentiation and find its possible molecular mechanisms for anti-osteoporosis.Methods OCT-1 cells were cultured with common methods.While growing well,cells were cultured with 3 doses(10 μg·mL-1,30u μg·mL-1 and 60 μg·mL-1)of Isopsoralen for 48 h,or with purified bone morphogenetic protein 2(BMP2)protein(50 ng·mL-1).We first determined the effect of Isopsoralen on cell proliferation by MTT assay.The real time RT-PCR was also used to quantify changes in the mRNA levels of several genes,such as BMP2,Runt-related transcription factor 2(Runx2),and Osterix(Osx).We also used the Western blot analysis to evaluate the expression of Runx2 and Osx proteins.At last we used the BMP2loxp/loxp mice to isolate the primary calvaria osteoblasts,cultured with Isopsoralen of the best dose for 48 h after the in vitro conditional gene knockout technology,and tested the gene expressions of Runx2 and Osx.And the alkaline Phosphatase(ALP)staining was also performed.Result Isopsoralen(10 μg·mL-1)can promote osteoblast proliferation obviously.From the real time RT-PCR analysis,Isopsoralen can enhance the BMP2 mRNA levels,the effect of 10 μg·mL-1 was the best,and 30 μg·mL-1 followed.In addition,we found that Isopsoralen(10 μg·mL-1)can enhance the Runx2 mRNA levels significantly.We also found that lower doses of Isopsoralen can enhance the Osx mRNA levels,the effect of 30 μg·mL-1 was the best,and 10 μg·mL-1 followed.From the Western blot analysis,low doses of Isopsoralen(10 μg·mL-1 and 30 μg·mL-1)can stimulate the expression of Runx2 protein.Besides,three doses of Isopsoralen can stimulate the expression of Osx protein,and the effect of 10 μg·mL-1 and 30 μg·mL-1 are better.Finally,the results of in vitro conditional gene knockout experiment showed that the overexpression of Runx2 and Osx genes in osteoblasts,as well as ALP staining,induced by Isopsoralen are BMP2 dependent.Conclusions In this study,we firstly demonstrate that Isopsoralen can stimulate osteoblast proliferation and differentiation by mediating BMP2/Runx2/Osx signaling pathway.

Comparative study of the effects of intramedullary nail fixation and minimally invasive percutaneous plate internal fixation technique on platelet activation and serum transforming growth factor-β1(TGF-β) 1 and bone morphogenetic protein-2 (BMP-2) in patients with tibial and fibular fracture / 中国骨伤

OBJECTIVE@#To investigate the effect of intramedullary nail fixation (IMN) and minimally invasive percutaneous plate internal fixation (MIPPO) techniques on tibiofibular fractures and their effect on platelet activation and serum transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2).@*METHODS@#Total of 105 patients with tibiofibular fractures from February 2019 to February 2020 were selected and divided into 53 cases in the MIPPO group and 52 cases in the IMN group. There were 29 males and 24 females with an average age of (41.74±6.05) years old in MIPPO group;in IMN group, 31 males and 21 females with an average age of (40.59±5.26) years old. The perioperative surgical indexes, postoperative complications, ankle function recovery at 12 months postoperatively, platelet activation indexes at 3 and 7 days preoperatively and postoperatively, and serum TGF-β1 and BMP-2 levels at 4 and 8 weeks preoperatively and postoperatively were compared between the two groups.@*RESULTS@#The operating time and fracture healing time in the MIPPO group were shorter than those in the IMN group(P<0.05); Compared with the preoperative period, the levels of GMP-140, PAC-1, CD63, and CD61 increased in both groups at 3 and 7 days after surgery, but were lower in the MIPPO group than in the IMN group(P<0.05);the levels of serum TGF-β1 and BMP-2 increased in both groups at 4 and 8 weeks after surgery compared with the preoperative period, and the postoperative complication rate in the MIPPO group was lower than that in the IMN group(P<0.05);the difference was not statistically significant in the excellent rate of ankle function recovery at 12 months follow-up after surgery between two groups(P>0.05).@*CONCLUSION@#Both intramedullary nail fixation and MIPO technique for treatment of tibia and fibula fractures can improve ankle joint function, but the latter has the advantages of short operation time, fast fracture healing, fewer complications, and light platelet activation. Serum TGF-β1, BMP-2 level improves quickly.

Influence of photobiomodulation and tricalcium silicate-based cement on viability and dentinogenic activity of cultured human dental pulp stem cells / Influência da fotobiomodulação e cimento de silicato tricálcio na viabilidade e atividade dentinogênica de células tronco cultivadas da polpa de dente humano

Objective: To assess the effect of application of Biodentine (BD), Photobiomodulation (PBM) using 810 nm diode laser and both on the proliferation and odontogenic differentiation of human dental pulp stem cells (HDPSCs). Material and Methods: HDPSCs were collected, isolated, and characterized and then divided into six groups: groups 1, control; groups 2, biodentine (BD); group 3, irradiation at 1 J/cm 2 of 810-nm diode laser; group 4, irradiation at 1 J/cm 2 and culture with BD; group 5, irradiation at 2 J/cm 2, and group 6, irradiation at 2 J/cm 2 and culture with BD. Viability assay was measured through MTT assay and Alkaline phosphatase (ALP) enzyme activity and mRNA levels of RUNX2, collagen 1 (Col-1) and BMP2 were also assessed. Results: Photobiomodulation at 1 and 2 J/cm 2 combined with biodentine significantly promoted HDPSCs proliferation (in MTT assay results) and odontogenic differentiation (through the gene expression of RUNX2, Col-1 and BMP2 levels (p < 0.05). Conclusion: Photobiomodulation at 2 J/cm 2 combined with biodentine enhanced proliferation and odontogenic differentiation of cultured HDPSCs and thus could further be beneficial for dentin regeneration (AU)

Objetivo: Avaliar o efeito da aplicação de Biodentina (BD), Fotobiomodulação (PBM) usando diodo de laser de 810 nm e ambos na proliferação e diferenciação odontogênica de células tronco cultivadas da polpa dental (HDPSCs). Material e Métodos: HDPSCs foram coletadas, isoladas, caracterizadas e então divididas em seis grupos: grupo 1, controle; grupo 2, biodentina (BD); grupo 3, irradiação com diodo de laser a 1 J/cm2 de 810- nm; grupo 4, irradiação a 1 J/cm 2 e cultivo com BD; grupo 5, irradiação a 2 J/cm2, e grupo 6, irradiação a 2 J/cm2 e cultivo com BD. A viabilidade foi mensurada através do teste MTT e a atividade da enzima Fosfatase alcalina (ALP), e níveis de RNAm de RUNX2, de colágeno 1 (Col-1) e de BMP2 foram também mensurados. Resultados: Fotobiomodulação a 1 e 2 J/cm 2 combinada com biodentina promoveu significativa proliferação de HDPSCs (nos resultados do teste MTT) e diferenciação odontogênica (através da expressão genética dos níveis de RUNX2, Col-1 e BMP2 (p < 0.05)). Conclusão: Fotobiomodulação a 2 J/cm2 combinada com biodentina aumentou a proliferação e diferenciação odontogênica de HDPSCs cultivadas e dessa forma poderia ser benéfica para a regeneração dentinária. (AU)

Horizontal ridge augmentation of a single atrophic site in the anterior maxilla using hydroxyapatite and rhBMP-2 / Augmento óseo horizontal de reborde alveolar en sitio atrófico único en maxila anterior usando hidroxiapatita y rhBMP-2

SUMMARY: Bone morphogenetic protein (rhBMP-2) is a powerful osteo-inductive growth factor widely used in bone reconstruction and both the vehicle used to administer it and the scaffold substrate could determine its success in clinical situations. The aim was to analyse the clinical behaviour of dental implants placed in single alveolar ridges with a horizontal deficiency in the maxillary anterior region that were reconstructed horizontally with rhBMP-2 and porous hydroxyapatite (HA). Inclusion criteria were both males and females, between the ages of 18 and 29 with single tooth loss of one upper incisor. Cone Beam Computed Tomography (CBCT) was used to take measurements prior to bone augmentation and again prior to the implant insertion. Surgery was carried out under local anaesthetic. In the primary procedure, bone substitute was introduced using porous HA and rhBMP-2; after 4 to 5 months, dental implant surgery was carried out and the implant placed; after 3 months of consolidation the provisional prosthesis was placed and then a definitive restoration was placed. Variables were analysed using the t-test with a p-value of < 0.05 in order to assess statistical significance. Thirteen subjects were included (6 females and 7 males). Bone augmentation resulted in a bone gain of 4.15mm (p=0.023), which was shown to be statistically significant. All of the grafts placed were successful and 13 implants were placed, using torques between 30 and 70N, without complications. For the final prostheses, 11 were screw retained and 2 were cemented in place. The horizontal bone augmentation using HA and rhBMP-2 is an efficient technique for single bone defects in the anterior maxillary area; clinical trials on a larger scale are needed to confirm these results.

RESUMEN: La proteína ósea morfogenética (BMP-2) es un potente osteoinductor utilizado ampliamente en técnicas reconstructivas; el vehículo de instalación es determinante en su evolución. El objetivo fue analizar el comportamiento clínico de implantes dentales instalados en rebordes alveolares únicos con deficiencia horizontal del sector anterior reconstruida horizontalmente con BMP-2 e hidroxiapatita (HA) porosa. Fueron incluidos sujetos de ambos sexos de entre 18 y 29 años, con pérdida dentaria unitaria a nivel de incisivos superiores. Se utilizó tomografía computadorizada para realizar mediciones en las etapas previa a la instalación del injerto y previo a la instalación del implante. Las cirugías fueron realizadas bajo anestesia local. En la primera intervención se realizó la instalación del injerto óseo utilizando HA porosa y BMP-2; después de 4 a 5 meses se realizó la instalación del implante dental; 3 meses después se realizó la conexión protésica y rehabilitación final. Las variables fueron estudiadas con la prueba t test considerando el valor de p< 0,05 para considerar significancia estadística. Trece sujetos fueron incluidos (6 mujeres y 7 hombres); con la reconstrucción ósea se obtuvo una ganancia ósea de 4,15mm (p=0.023) que fue estadísticamente significativo. No existió pérdida en ningún injerto realizado; se instalaron 13 implantes con torques entre 30 y 70N sin complicaciones; se realizaron prótesis fijas atornilladas en 11 casos y cementadas en 2 casos. La técnica con HA y BMP- 2 es eficiente para reconstruir defectos horizontales en perdidas unitarias del sector anterior maxilar; ensayos clínicos de mayor escala son necesarios para confirmar estos resultados.

Bioactivity of the peptide P17-bone morphogenetic protein-2/nano-hydroxyapatite-mineralized collagen composite / 中国组织工程研究

BACKGROUND: A mineralized collagen composite, i.e. nano-hydroxyapatite/collagen (nHAC) has biomimetic three-dimensional structure and good bioactive properties. As a bone tissue engineering material, it is widely used in bone defect repair. A newly designed P17-bone morphogenetic protein-2 (P17-BMP2) has good biocompatibility and osteogenic capacity. Therefore, the composite scaffold material was prepared by combining the new P17-BMP-2 and nHAC, which might be used for the enhancement of osteogenic capacity in the treatment of bone defects. OBJECTIVE: To investigate the bioactivity of the P17-BMP-2/nHAC composite. METHODS: Rabbit bone marrow mesenchymal stem cells were seed on the P17-BMP-2/nHAC composite and nHAC. After 3 and 7 days of culture, the relative expression level of alkaline phosphatase was detected by RT-PCR. The subcutaneous implantation of P17-BMP-2/nHAC (experimental group) and nHAC (control group) into Sprague-Dawley rats was performed. Masson staining was performed for histological analysis at 12 and 35 days of implantation. P17-BMP-2/nHAC (experimental group) and nHAC (control group) were implanted into the white rabbit mandibular box-shaped bone defect, respectively. At 5 and 15 weeks, gross observation and X-ray were performed. The study was approved by the Medical Ethics Committee of China Medical University School & Hospital of Stomatology. RESULTS AND CONCLUSION: (1) The relative expression level of alkaline phosphatase in the P17-BMP-2/nHAC group was significantly higher than that in the nHAC group (P < 0.05). (2) The result of subcutaneous implantation showed that the acute inflammatory response initiated by the P17-BMP-2/nHAC or nHAC was not found. More activated fibroblasts growing into the implants could be found on the sections of P17-BMP-2/nHAC compared to that of nHAC at 35 days after implantation. (3) In the bone defect repair test, gross observation showed that both materials held good defect repair ability, the defect area began to reduce at 5 weeks after implantation, and the defect surface became flat at 15 weeks after implantation. X-ray examination showed that compared with the control group, the defect area was more significantly reduced in the experimental group. (4) These results indicate that P17-BMP-2/nHAC composite scaffold has higher bioactivity and a stronger ability to repair bone defect.

Results of the bone healing performance of Osteo-calcium 5 on based preparation tested by internal fixation surgery patients / Шинэ санаа Шинэ нээлт

Background@#Even though there are lots of good treatment methods, medicines and biomaterials in use for broken bones due to international scientific researches on the ossifying process and its mechanism, which are being studied on high level and bringing new solutions, there is still a lack of inexpensive but good quality medicines that have low side effects and specifically targets on the enhancement of osteoblasts. </br> Despite of the level of fractures, therapeutic effects and multiple bone fractures, only certain drugs and medicines such as solution of baragshun and rhodiola, kitchen grime, chili-san and calcium are used for ossifying broken bones and fractures in Mongolia which indicates that there are certain needs of new medicine and drugs.@*Methods@#Patients who had a intramedullary osteosynthesis surgery to deal with tibia bone fractures were divided randomly into two groups; 1.5 gram Osteo calcium-5 is given to the experimental group while the comparison group got Calcium nycomed D3 for 60 days. </br> X-ray pictures were taken at the 56th and the 84th day after the fractures were diagnosed and analyzed by Warden method (et al 2009). TGF-β1, BMP-2 cytokines and alkaline phosphatase were evaluated on the 3rd, the 14th and the 42nd day of the study.@*Results@#Compared to the comparison group, which used Calcium Nycomed D3, the experimental group patient, who used osteocalcium-5, had a similar TGF-β1 level at the 3rd day (p=1.0), a higher level at the 14th day (p=0.0001) and a lower level at the 42nd day (p=0.015).Analyzing fracture ossifying process with the Warden et al method shows that the ossifying points of the Osteo calcium-5 group were 1.73 on 53rd day and 2.79 on 84th day while the ossifying points of the comparison group was 1.41 on 56th day and 2.35 on 84th day (p=0.034),( p=0.04).@*Conclusions@#According to the study, Osteo calcium-5 has proven to have better effects on decreasing inflammation process and accelerating bone ossify, cartilage and bone formation with the analysis of cytokine in serum and structure.

The regulatory mechanisms of IGF1 in the osteogenic differentiation of canine MSMSCs via BMP2⁃Smad 1/5 signaling pathway / 口腔疾病防治

Objective@#To investigate the role of the bone morphogenetic protein 2 (BMP2)⁃Smad1/5 and p38MAPK signaling pathways in the osteogenic differentiation of MSMSCs by insulin⁃like growth factor 1 (IGF1).@* Methods @#A re⁃ combinant adenovirus (RAD) and IGF1 expressing IGF1 gene were constructed. After osteogenic induction, qRT⁃PCR and Western blot were used to detect the phosphorylation level of Smad1/5 and the expression of the BMP⁃2 protein in the BMP⁃Smad signaling pathway; immunohistochemistry was used to observe the nuclear translocation of Smad1/5; qRT⁃PCR and Western blot were used to detect IGF with Noggin and SB203580, inhibitors of the p38MAPK signaling path⁃ way 1⁃mediated osteogenic differentiation of MSMSCs@* Results@#The recombinant IGF1 adenovirus was constructed suc⁃ cessfully. MSMSCs were cultured in inductive medium after infection with different concentrations of Ad⁃IGF1, and then, the protein levels of BMP2 and p⁃Smad1/5 increased. IGF1 can also induce nuclear translocation of Smad1/5. In addition, Noggin significantly reduced the phosphorylation level of Smad1/5 and the formation of mineralized nodules in the MSMSCs. The mRNA levels of Runx2, OPN and ALP also decreased. In contrast, SB203580 decreased neither the phosphorylation level of p38 nor the mRNA expression of Runx2, OPN and ALP in the Ad⁃IGF1 MSMSCs@* Conclu⁃sion@#IGF1 can promote the osteogenic differentiation of MSMSCs via the BMP2⁃Smad1/5 signaling pathway. In con⁃ trast, IGF1 may not promote the osteogenic differentiation of MSMSCs via the p38MAPK signaling pathway.

Bone morphogenetic proteins 2, 6, and 9 differentially regulate the osteogenic differentiation of immortalized preodontoblasts

Our study attempted to compare the efficacies of bone morphogenetic protein (BMP) 2, 6, and 9 in inducing osteogenic differentiation of preodontoblasts (PDBs). We immortalized PDBs by introducing a reversible SV40 T antigen-based immortalization system. Cell proliferation capability was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The effects of BMP2, 6, and 9 on the osteogenic differentiation of immortalized preodontoblasts (iPDBs) were measured by alkaline phosphatase (ALP) activity assays and alizarin red S staining. The expression of osteogenic markers was evaluated by semiquantitative real-time polymerase chain reaction analysis. To assess ectopic bone formation, rat-derived iPDBs were transfected in culture with adenoviral vectors designated Ad-BMP2, 6, and 9 and subcutaneously or intramuscularly injected into mice. Several BMPs retained endogenous expression in PDBs and regulated the mRNA expression of mineralized tissue-associated proteins. ALP activity and mineralized nodule formation were significantly increased in the Ad-BMP9-transfected group relative to the control group. In addition, the most significant hard tissue formation was in this group. The results indicated that BMP signaling was involved in the osteogenic differentiation of iPDBs. BMP9 could be an efficacious accelerant of the osteogenic differentiation of iPDBs.

Rational design of type-IA receptor-derived cyclic peptides to target human bone morphogenic protein 2

Human bone morphogenetic protein 2 (BMP2) is a bone-growth regulatory factor involved in the formation of bone andcartilage, and has been recognized as an attractive therapeutic target for a variety of bone diseases and defects. Here, wereport successful design of a head-to-tail cyclic peptide based on crystal structure to target BMP2. Computational alaninescanning identifies two hotspot regions at the crystal complex interface of BMP2 with its type-IA receptor; promising one isstripped from the interface to derive a linear self-inhibitory peptide RPS2[r78-94] that covers residues 78–94 of the receptorprotein. Dynamics simulation and energetics analysis reveal that the peptide is highly flexible in isolated state and cannotspontaneously bind to BMP2. The RPS2[r78-94] peptide is further extended from its N- and C-termini until reaching twospatially vicinal residues 74 and 98 in the crystal structure of intact BMP2–receptor complex system, consequently resultingin a longer peptide RPS2[r74-98], which is then cyclized in a head-to-tail manner to obtain its cyclic counterpartcycRPS2[r74-98]. Computational analysis suggests that the cyclic peptide can well maintain in a conformation similar withits active conformation in complex crystal structure, exhibiting a smaller disorder and a larger potency than its linearcounterpart. Further assays confirm that the two linear peptides RPS2[r78-94] and RPS2[r74-98] are nonbinders of BMP2,whereas, as designed, the cyclic peptide cycRPS2[r74-98] can bind to BMP2 with a moderate affinity. The cyclic peptide isexpected as a lead molecular entity to develop new and potent peptide-based drugs for BMP2-targeted therapy.

Pilose antler aqueous extract promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by stimulating the BMP-2/Smad1, 5/Runx2 signaling pathway / 中国天然药物

Peptides from Pilose antler aqueous extract (PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE's effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction (Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200 μg·mL showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.

In Situ Gelling Hydrogel with Anti-Bacterial Activity and Bone Healing Property for Treatment of Osteomyelitis

BACKGROUND: Despite the development of progressive surgical techniques and antibiotics, osteomyelitis is a big challenge for orthopedic surgeons. The main aim of this study is to fabricate an in situ gelling hydrogel that permits sustained release of antibiotic (for control of infection) and growth factor (for induction of new bone formation) for effective treatment of osteomyelitis. METHODS: An in situ gelling alginate (ALG)/hyaluronic acid (HA) hydrogel containing vancomycin (antibiotic) and bone morphogenetic protein-2 (BMP-2; growth factor) was prepared by simple mixing of ALG/HA/Na₂HPO₄ solution and CaSO₄/vancomycin/BMP-2 solution. The release behaviors of vancomycin and BMP-2, anti-bacterial effect (in vitro); and therapeutic efficiency for osteomyelitis and bone regeneration (in vivo, osteomyelitis rat model) of the vancomycin and BMP-2-incorporated ALG/HA hydrogel were investigated. RESULTS: The gelation time of the ALG/HA hydrogel was controlled into approximately 4 min, which is sufficient time for handling and injection into osteomyelitis lesion. Both vancomycin and BMP-2 were continuously released from the hydrogel for 6 weeks. From the in vitro studies, the ALG/HA hydrogel showed an effective anti-bacterial activity without significant cytotoxicity for 6 weeks. From an in vivo animal study using Sprague-Dawley rats with osteomyelitis in femur as a model animal, it was demonstrated that the ALG/HA hydrogel was effective for suppressing bacteria (Staphylococcus aureus) proliferation at the osteomyelitis lesion and enhancing bone regeneration without additional bone grafts. CONCLUSIONS: From the results, we suggest that the in situ gelling ALG/HA hydrogel containing vancomycin and BMP-2 can be a feasible therapeutic tool to treat osteomyelitis.

Zinc upregulates bone-specific transcription factor Runx2 expression via BMP-2 signaling and Smad-1 phosphorylation in osteoblasts / 한국영양학회지

PURPOSE: Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression. METHODS: Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn− (1 µM Zn) or Zn+ (15 µM Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed. RESULTS: In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn−. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn−. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn−, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn−, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn−. CONCLUSION: The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.

The effects of He-Ne laser on the expression of extracellular matrix and its regulatory factors in degenerated temporomandibular joint of rabbits / 实用口腔医学杂志

Objective: To observe the effects of He-Ne laser on the expression of extracellular matrix and its regulatory factors in degenerated temporomandibular joint(TMJ) of rabbits. Methods: 40 New Zealand adult white rabbits were randomly divided into normal group,sham model group,TMJOA model group and laser treatment group(n = 10),the rats in treatment group were treated by He-Ne laser irradination at acupoints. The rats in each group were divided into 1 d and 11 d groups(n = 5). The animals were respectively killed 1 d and 11 d after operation and HE staining was used to observe the histomorpholy. The protein expression of extracellular matrix and its regulatory factors were examined by Western blot. Results: After He-Ne laser treatment,the fiber layer of condylar cartilage was slightly loose,part of the fiber was newly produced. The level of Col-2,PRG-4,TIMP-1,BMP-2 was up-regulated and the of MMP-13 was down-regulated in the 11 d treatment group. Conclusion: He-Ne laser irradiation on acupoints may up-regulate the expression of extracellular matrix (Col-2 and PRG-4) and its regulatory factors (TIMP-1 and BMP-2),down-regulate the expression of MMP-13.

Effects of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) release from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone

Multiple growth factors can be administered to mimic the natural process of bone healing in bone tissue engineering. We investigated the effects of sequential release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) from polylactide-poly (ethylene glycol)-polylactide (PELA) microcapsule-based scaffolds on bone regeneration. To improve the double emulsion/solvent evaporation technique, VEGF was encapsulated in PELA microcapsules, to which BMP-2 was attached. The scaffold (BMP-2/PELA/VEGF) was then fused to these microcapsules using the dichloromethane vapor method. The bioactivity of the released BMP-2 and VEGF was then quantified in rat mesenchymal stem cells (rMSCs). Immunoblotting analysis showed that BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblasts via the MAPK and Wnt pathways. Osteoblast differentiation was assessed through alkaline phosphatase expression. When compared with simple BMP-2 plus VEGF group and pure PELA group, osteoblast differentiation in BMP-2/PELA/VEGF group significantly increased. An MTT assay indicated that BMP-2-loaded PELA scaffolds had no adverse effects on cell activity. BMP-2/PELA/VEG promoted the differentiation of rMSCs into osteoblast via the ERK1/2 and Wnt pathways. Our findings indicate that the sequential release of BMP-2 and VEGF from PELA microcapsule-based scaffolds is a promising approach for the treatment of bone defects.

Polylactic/polyglycolic acid copolymer is a good carrier for bmp-2 on bone regeneration? / Copolímero do ácido polilático/poliglicólico é um bom carreador para bmp-2 na regeneração óssea?

The aim of this work was to evaluate the role of polylactic /polyglycolic acid copolymer as carrier for BMP-2 on bone regeneration in rat calvarium. Forty five adult male rats underwent 5-mm critical defects in bone calvaria to be divided into three groups according to the filling materials: Control-blood clot; PLGA-polylactic acid Polyglycolic/copolymer; PLGA + BMP-2 -polylactic acid Polyglycolic/copolymer associated with BMP-2. Sacrifice of animals occurred at 5, 15 and 30 days after surgery The evaluation of new bone formation was obtained by histomorphometry, while OPG and RANKL proteins were observed by immunohistochemistry. Statistical analysis was performed by means of nonparametric tests based on quantitative variables of independent samples. Considering the amount of newly formed bone, significant difference was detected between PGLA (178,2±137,5µm) and the other groups, at day 30. In PLGA + BMP-2 and control groups, the expression of RANKL was prevalent on the OPG in the periods of 15 and 30 days, suggesting a favorable condition for bone reabsorption in these periods. Therefore, immunoexpression of RANKL and OPG and bone formation observed in different groups and periods of analysis showed that the polylactic/polyglycolic acid copolymer does not act as a good carrier for BMP-2.

O objetivo deste estudo foi avaliar o copolímero do ácido polilático/poliglicólico como carreador para BMP-2 na regeneração óssea da calvária de ratos. Foram utilizados 45 ratos adultos machos. Defeitos ósseos críticos de 5mm de diâmetro foram realizados com uma broca trefina na calvária dos animais. Os animais foram divididos em três grupos, de acordo com o material de notas para preenchimento dos defeitos: Controle - coágulo; PLGA - copolímero ácido polilático/poliglicólico; PLGA + BMP-2 - copolímero do ácido polilático/poliglicólico associado a BMP-2. O sacrifício ocorreu aos 5, 15 e 30 dias após a cirurgia. A avaliação da neoformação óssea foi obtida por histomorfometria, enquanto a análise de marcação para as proteínas OPG e RANKL foi observada por imunohistoquímica. Análises estatísticas foram realizadas por meio de testes não paramétricos de variáveis quantitativas em amostras independentes. Com relação à quantidade de tecido ósseo neoformado, observou-se diferença estatística significante entre o grupo PLGA (178,2±137,5µm) e os demais, no período de 30 dias. Nos grupos PLGA+BMP-2 e Controle, a marcação de RANKL foi predominante sobre a marcação de OPG nos períodos de 15 e 30 dias, evidenciado uma condição favorável para a reabsorção óssea nestes períodos. Portanto, a marcação de RANKL e OPG, e a formação óssea observada nos diferentes grupos e tempos de análise mostrou que o copolímero de ácido polilático/poliglicólico não atua como um bom carreador para BMP-2.

Early changes in retinal structure and BMP2 expression in the retina and crystalline lens of streptozotocin-induced diabetic pigs / 한국실험동물학회지

PURPOSE: This study aims to evaluate early changes in retinal structure and BMP2 expression in the retina and crystalline lens by comparing streptozotocin-induced diabetic pigs and normal control group pigs. METHODS: Five eye samples from five diabetic Micro-pigs (Medikinetics, Pyeongtaek, Korea) and five eye samples from five control pigs bred in a specific pathogen-free area were used. Diabetes was developed through intravenous injection of nicotinamide and streptozotocin, and the average fasting glucose level was maintained at 250 mg/dL or higher for 16 weeks. To evaluate BMP2 expression in the retina and crystalline lens, Western blotting was performed. RESULTS: In Hematoxylin and Eosin staining, most diabetic pigs showed structural abnormalities in the inner plexiform layer. The number of nuclei in the ganglion cell layer within the range of 10⁴µm² was 3.78±0.60 for diabetic pigs and 5.57±1.07 for control group pigs, showing a statistically significant difference. In immunohistochemical staining, diabetic retinas showed an overall increase in BMP2 expression. In Western blotting, the average BMP2/actin level of diabetic retinas was 1.19±0.05, showing a significant increase compared to the 1.06±0.03 of the control group retinas (P=0.016). The BMP2/actin level of diabetic crystalline lenses was similar to the control group crystalline lenses (P=0.730). CONCLUSIONS: Compared to control group pigs, the number of nuclei in the inner nuclear layer of retinas from streptozotocin-induced diabetic pigs decreased, while an increase in BMP2 expression was observed in the retina of diabetic pigs.

The effects of β tricalcium phosphate/collagen scaffold loaded with human bone morphogenetic protein 2 plasmid on the osteogenesis ability of MC3T3-E1 cells / 实用口腔医学杂志

Objective:To study the effects of beta tricalcium phosphate(β-TCP)/collagen scaffold loaded with human bone morphogenetic protein 2 (hBMP2) plasmid on the osteogenesis ability of MC3T3-E1 cells.Methods:hBMP2 DNA plasmid-modified β-TCP/collagen scaffold and the naked plasmid(control) were constructed.MC3T3-E1 cells were respectively in vitro cultured onto the β-TCP/collagen scaffold with hBMP2(Z) and with control plasmid(Z0),on peace dish with the saffold and hBMP2(M) and with the control plasmid(M0).The surface morphology of the samples was observed by SEM.Osteogenesis of the cells was examined by alkaline phosphatase activity(ALP) test,real-time fluorescent quantitative PCR for the detection of Runx2,OCN,ALP and OPN mRNA expression.Data were statistically analyzed.Results:The composite sample surface of plasmid DNA containing hBMP2 modified β-TCP/collagen was porous;group Z and M showed highter ALP activity and higher mRNA expression of Runx2,OCN,ALP and OPN than group Z0 and M0;so did group Z than group M.Conclusion:Porous β-TCP/collagen scaffold loaded with BMP2 DNA is potential for osteoinduction.

Recombinant Human Bone Morphogenic Protein-2 (BMP-2): A Newer & Novel Osteoinductive Treatment Modality for Non-union of Bones.

Background: The non-union of bones is a multifactorial phenomenon. In this study, it was emphasized to evaluate the efficacy and safety of bone morphogenetic protein-2 (BMP-2) as a bone-stimulating agent in the treatment of non-unions. Methods: Fifteen patients [5 males, mean age 51.06 years (range: 21—75)] with sixteen non-unions were treated with BMP-2. There were eleven femoral non-union, three humerus, one ulna, one distal fibula non-union. The mean follow-up was 22.06 months. Results: Both clinical and radiological union occurred in 15 (93.75%) non unions cases. Radiological union achieved within a mean time of 15.75 weeks. The remaining one show incomplete union with recalcitrant formation was asymptomatic and having good pain free range of movement, declines further intervention. No complications or adverse effects from the use of BMP-2 were encountered. Conclusion: In this study, it was observed that BMP-2 is a powerful adjunct and one of the safe armamentarium for the surgeon to handle difficult and challenging clinical conditions.