与胶原特异性结合的BMP2模拟肽/PLGA 3D打印复合支架的制备及成骨诱导活性

将胶原绑定结构域(CBD)多肽序列与骨形态发生蛋白2模拟肽(BMP2-MP)序列连接制备具有胶原绑定能力的CBD-BMP2-MP, 再将CBD-BMP2-MP与聚丙交酯-乙交酯/胶原(PLGA/COL)3D打印支架相结合, 以支架表面的胶原成分为媒介, 将CBD-BMP2-MP更有效地固定于骨修复材料上, 达到对其进行改性的目的. 利用扫描电子显微镜(SEM)、 电子万能试验机和接触角测量仪对复合支架表面形貌、 力学强度和亲水性等材料学性能进行评价. 用荧光成像法评测 CBD-BMP2-MP及BMP2-MP与支架材料的结合能力. 在各组支架材料表面接种MC3T3-E1细胞进行体外培养, 采用CCK-8、 鬼笔环肽荧光染色、 茜素红染色及qPCR综合评价细胞在材料表面的黏附、 增殖和成骨分化等细胞行为, 研究CBD-BMP2-MP修饰的3D多孔PLGA/COL复合支架的生物学性能. 研究结果表明, 利用3D打印技术制备的多孔支架具有形貌可控的孔隙结构, 为细胞生长创造更有利的细胞微环境, 支架表面胶原成分的加入提高了支架材料的亲水性, 同时对支架材料本身的力学性能无任何影响, 提高了复合支架本身的生物相容性. 与普通BMP2-MP相比, CBD-BMP2-MP具有更好的胶原绑定能力, 与复合支架的结合更稳定, 提高了PLGA/COL复合支架对BMP2-MP的负载能力. 支架表面负载CBD-BMP2-MP后具有极强的促细胞成骨分化能力. MC3T3-E1细胞表现出更高的钙沉积能力, 并且成骨分化相关基因Runx2, ALP, COL-I及OPN等水平也有了明显提升. 表明CBD-BMP2-MP多孔复合支架具有良好的生物相容性和成骨诱导活性, 在骨组织修复领域具有良好的应用前景.     

The pharmacological effects of morroniside and loganin isolated from Liuweidihuang Wan, on MC3T3-E1 cells

Liuweidihuang wan (LW), initially a well-known formula for curing "wu chi wu ruan", is commonly used nowadays for clinical treatment of postmenopausal osteoporosis (PO), but the identity of the effective substance(s) remains unclear. The present study was designed to evaluate the effects of morroniside and loganin isolated from LW on the proliferation, differentiation and apoptosis of MC3T3-E1 cells, as well as the possible mechanism of action. Morroniside and loganin had no effects on the proliferation of MC3T3-E1 cells, but both susbtances could improve the activity of alkaline phosphatase (ALP), and increase the contents of collagen type I and osteocalcin. Simultaneously, the mRNA expression of caspase-3, capase-9, RANKL was down-regulated and that of bcl-2 was up-regulated, which partially explains the anti-osteoporosis mechanism in MC3T3-E1 cells. In conclusion, morroniside and loganin may directly promote the differentiation and inhibit the apoptosis of MC3T3-E1 cells, and accordingly indirectly reduce bone resorption, which makes them promising natural drugs leads for treating PO in the near future.     

硅表面CS/BSA复合微图形的制备及表征

采用微加工方法在硅表面成功地制备出壳聚糖/牛血清白蛋白(CS/BSA)复合微图形. 借助光镜和荧光显微镜对复合微图形进行形貌分析. 利用大肠杆菌和白色葡萄球菌定量考察了CS/BSA复合微图形的抗菌性能, 结果表明, 硅表面沟槽交叉状CS/BSA复合微图形对两种细菌都具有较好的抗菌效果. 通过MC3T3-E成骨细胞培养考察了复合微图形的细胞相容性, 结果表明, CS/BSA复合微图形对于细胞的生长方向具有较强的诱导性, 可促进细胞在材料表面的黏附、铺展及增殖分化. 结果表明, 采用微转移模塑法制备的CS/BSA复合微图形具有较好的抗菌性和细胞相容性.     

Effects of combined mechanical stimulation on the proliferation and differentiation of pre-osteoblasts

We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10 % strain for cyclic strain and 1.0 MHz and 30 mW/cm(2) for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation.     

Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells

Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.     

Regulation of cellular responses to macroporous inorganic films prepared by the inverse-opal method

Regenerative medicine for repairing damaged body tissues has recently become critically important. Cell culture scaffolds are required for the control of cell attachment, proliferation, and differentiation in in vitro cell cultures. A new strategy to control cell adhesion, morphology, and proliferation was developed by culturing mouse osteoblast-like MC3T3-E1 cells on novel cell culture scaffolds fabricated using ordered nanometer-sized pores (100, 300, 500, and 1000 nm). Results of this study indicate that after 72 h of incubation, the number of cells cultured on a silica film with a pore size of 1000 nm was similar to or slightly lower than that cultured on a non-porous control silica film. Films with 100-500 nm pore sizes, however, resulted in the cell growth inhibition. Morphology of the cultured cells revealed increased elongation and the formation of actin stress fibers was virtually absent on macroporous silica films with 100-500 nm pore size. Vinculin molecules expressed in cells cultured on the non-porous silica films showed many clear focal adhesions, whereas focal contacts were insufficiently formed in cells cultured on macroporous films. The influence of hydroxyapatite (HAp) and alumina scaffolds on the behavior of MC3T3-E1 cells was also evaluated. The proliferation rate of MC3T3-E1 cells cultured on HAp films with 1000 nm pore size was increased to approximately 20% above than that obtained of cells cultured on non-porous HAp films. These results demonstrate that the pore size and constituents of films play a role in controlling the morphology and proliferation rate of MC3T3-E1 cells.     

Improvement of differentiation and mineralization of pre-osteoblasts on composite nanofibers of poly(lactic acid) and nanosized bovine bone powder

The effect of NBM incorporation in PLA nanofibers on their mechanical properties and the differentiation and mineralization of osteoblasts was studied. At 20% NBM, the Young's modulus of the nanofibers was 37.78 +/- 4.23, significantly larger than that of pure PLA nanofibers. MC3T3-E1 pre-osteoblasts attached to both types of nanofibers and developed full osteogenic phenotypes. A profound effect of NBM on the mineralization of MC3T3-E1 pre-osteoblasts was confirmed, suggesting that NBM/PLA composite nanofibers exhibit properties similar to those of the native collagen-rich mineralized bone matrix, and could therefore serve as a temporary substrate for facilitating the differentiation and mineralization of bone-forming cells.     

Emodin-8-O-β-D-glucoside from Polygonum amplexicaule D. Don var. sinense Forb. promotes proliferation and differentiation of osteoblastic MC3T3-E1 cells

Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a famous traditional herb used to treat fractures, rheumatoid arthritis, muscle injury and pain. The present study was designed to investigate a PAF derived-chemical compound emodin-8-O-β-D-glucoside (EG) on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro. A compound was isolated from PAF extract by HPLC and identified as emodin-8-O-β-D-glucoside (EG) by spectroscopic methods. EG significantly promoted cell proliferation at 0.1-100 ng/mL, and increased the cell proportion in S-phase from 16.34% to 32.16%. Moreover, EG increased alkaline phosphatase (ALP) expression in MC3T3-E1 cells at the concentration from 0.1 to 100 ng/mL and inhibited PGE(2 )production induced by TNF-α in osteoblasts at the concentrations ranging from 10-100 ng/mL, suggesting that cell differentiation was induced in MC3T3-E1 osteoblasts. Taken together, these results indicated compound EG directly stimulated cell proliferation and differentiation of osteoblasts, therefore this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.     

HPMC crosslinked chitosan/hydroxyapatite scaffolds containing Lemongrass oil for potential bone tissue engineering applications

The chitosan (CS), hydroxypropyl methyl cellulose (HPMC), hydroxyapatite (HAp and Lemon grass oil (LGO) based scaffolds was prepared by freeze gelation method. The composite formation was confirmed by FTIR (Fourier-transform infrared spectroscopy) analysis and surface morphology was evaluated by SEM (Scanning Electron Microscopy) analysis. The mechanical strength, biodegradation, swelling, porosity and antibacterial activity were evaluated on the basis of LGO contents. The scaffold structure was porous and the mechanical strength was enhanced as a function of LGO contents. The scaffold properties analysis revealed the biodegradation nature and swelling behavior of CS-HPMC-HAp-LGO was also affected significantly as a function of LGO contents. The cytotoxicity of CS-HPMC-HAp-LGO was studied against MC3T3-E1 cells and based on cell viability, no toxic sign was observed. The antimicrobial activity was evaluated against S. aureus and CS-HPMC-HAp-LGO scaffolds showed promising activity, which was varied as a function of LGO contents. The findings revealed that the CS-HPMC-HAp-LGO are biocompatible and have potential for bone tissue engineering.     

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS ⁿ ) was developed for screening of potential anti-osteoporosis agents in E.?koreanum. Four compounds identified as epimedin?A, epimedin?B, epimedin?C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in?vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS ⁿ is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.     

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MSⁿ) was developed for screening of potential anti-osteoporosis agents in E. koreanum. Four compounds identified as epimedin A, epimedin B, epimedin C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MSⁿ is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.

     

L-Arginine/nanofish bone nanocomplex enhances bone regeneration via antioxidant activities and osteoimmunomodulatory properties

In this study,a promising strategy has been developed to promote bone regeneration by combining antioxidant activities and osteoimmunomodulatory properties.Herein,an L-arginine/nanofish bone(Arg/NFB) nanocomplex has been prepared and evaluated in vitro and in vivo.The Arg/NFB nanocomplex possesses good antioxidant activities and could modulate the polarization of non-activated macrophage into different types and induce the secretion of pre-inflammato ry,anti-inflammatory,osteogenic as well as angiogenic cytokines.Additionally,the regulated immune microenvironment can enhance the osteogenic differentiation of mouse embryo osteoblast precursor cells(MC3 T3-E1) and angiogenic capacity of human umbilical vein endothelial cells(HUVECs),leading to the improved formation of mineralized nodules,alkaline phosphatase activity and angiogenic effects.In vivo results with cranial defect models reveal that the treatment of Arg/NFB nanocomplex exhibited significant improvement of new bone formation and angiogenesis.All the results demonstrate Arg/NFB nanocomplex with antioxidant activities and osteoimmunomodulatory properties could be a new idea for developing the next generation of bone regeneration biomaterials.     

Comparison of L-tryptophan binding capacity of BSA captured by a polymer brush with that of BSA adsorbed onto a gel network

A polymer brush containing a diethylamino group as an anion-exchange group was appended onto a polymer substrate by radiation-induced graft polymerization and subsequent chemical modifications. Bovine serum albumin as a chiral ligand for L-tryptophan was bound to the polymer brush at a density ranging from 17 to 150 g BSA/l. For comparison, BSA was adsorbed onto the gel network containing a diethylaminoethyl group. The molar binding ratio of L-tryptophan to BSA on the polymer brush was 1.7-fold higher than that to BSA on the gel network.     

Effect of phosphorylated organic compound on the adsorption of bovine serum albumin by hydroxyapatite.

The amount of adsorption of bovine serum albumin (BSA) by hydroxyapatite (HAP) increased with a concentration of CaCl2 due to the bridging effect of Ca2+ between adsorbate BSA and adsorbent HAP. On the other hand, it decreased remarkably with a concentration of K2HPO4. This was explained in terms of the effects of ionic strength and competitive adsorption between inorganic phosphate anion (Pi) and BSA, because BSA is in negatively charged over the examined pHs. A similar effect was observed in the presence of phosphorylated compounds such as phosphoserine, phytate, and phosphorylated polyvinylalcohol. The inhibiting effect of these compounds was stronger than that of their mother compounds (serine, inositol, and polyvinylalcohol). This result shows that phosphate groups bound to the mother compounds interfere with the adsorption of BSA by HAP in the same manner that Pi does. Although the adsorption of BSA was almost irreversible with respect to dilution with water, desorption was performed when these organic phosphorylated compounds were added after the accomplishment of the adsorption of BSA. However, the effective concentration of the phosphorylated compounds for the desorption of BSA was fairly higher than that for the competitive inhibition against the BSA adsorption.     

Probing protein adsorption onto mercaptoundecanoic acid stabilized gold nanoparticles and surfaces by quartz crystal microbalance and zeta-potential measurements

The adsorption characteristics of three proteins [bovine serum albumin (BSA), myoglobin (Mb), and cytochrome c (CytC)] onto self-assembled monolayers of mercaptoundecanoic acid (MUA) on both gold nanoparticles (AuNP) and gold surfaces (Au) are described. The combination of quartz crystal microbalance measurements with dissipation (QCM-D) and pH titrations of the zeta-potential provide information on layer structure, surface coverage, and potential. All three proteins formed adsorption layers consisting of an irreversibly adsorbed fraction and a reversibly adsorbed fraction. BSA showed the highest affinity for the MUA/Au, forming an irreversibly adsorbed rigid monolayer with a side-down orientation and packing close to that expected in the jamming limit. In addition, BSA showed a large change in the adsorbed mass due to reversibly bound protein. The data indicate that the irreversibly adsorbed fraction of CytC is a monolayer structure, whereas the irreversibly adsorbed Mb is present in form of a bilayer. The observation of stable BSA complexes on MUA/AuNPs at the isoelectric point by zeta-potential measurements demonstrates that BSA can sterically stabilize MUA/AuNP. On the other hand, MUA/AuNP coated with either Mb or CytC formed a reversible flocculated state at the isoelectric point. The colloidal stability differences may be correlated with weaker binding in the reversibly bound overlayer in the case of Mb and CytC as compared to BSA.     

In vitro Degradation and Biocompatibility of Ca-P Coated Magnesium Alloy

Calcium-phosphate compounds(Ca-P) coating was prepared on an Mg-Al alloy(AZ60). Biodegradation of Ca-P coated magnesium alloy was evaluated in simulated body fluid(SBF) by examining the changes in magnesium ion concentration and pH value, which indicated that the Ca-P coating on magnesium alloy strongly affected the corrosion of magnesium alloy. Osteoblast MC3T3-E1 cells were utilized to investigate the cellular cytocompatibility. The cytocompatibility was measured by carrying out a series of tests, such as cholecystokinin-octapeptide(CCK-8) test, alkaline phosphatase activity(ALP) test, cellular morphology of hematoxylin-eosin(HE) staining and the induction of apoptosis. It was found that the cell function showed better in the Ca-P coated Mg-alloy extract than in the uncoated magnesium alloy extract. In summary, the results indicate that the Ca-P coating can improve the corrosion resistance of magnesium alloy and elevate cellular proliferation and differentiation of osteoblast MC3T3-E1 cells.     

Microcontact printed antibodies on gold surfaces: function, uniformity, and silicone contamination

The function of microcontact printed protein was investigated using surface plasmon resonance (SPR) imaging, X-ray photoelectron spectroscopy spectroscopy (XPS), and XPS imaging. We chose to analyze a model protein system, the binding of an antibody from solution to a microcontact printed protein antigen immobilized to a gold surface. SPR imaging experiments indicated that the microcontact printed protein antigen was less homogeneous, had increased nonspecific binding, and bound less antibody than substrates to which the protein antigen had been physically adsorbed. SPR images of substrates contacted with a poly(dimethylsiloxane) stamp inked with buffer alone (i.e., no protein) revealed that significant amounts of silicone oligomer were transferred to the surface. The transfer of the silicone oligomer was not homogeneous, and the oligomer nonspecifically bound protein (BSA and IgG) from solution. XPS spectroscopy and imaging were used to quantify the amount of silicon (due to the presence of silicone oligomer), as well as the amounts of other elements, transferred to the surface. The results suggest that the silicone oligomer introduced by the printing process reduces the overall binding capacity of the microcontact-printed protein compared to physically adsorbed protein.     

Levan polysaccharide from Erwinia herbicola protects osteoblast cells against lipopolysaccharide-triggered inflammation and oxidative stress through regulation of ChemR23 for prevention of osteoporosis

Osteoblast cell injury is a type of degenerative disorder characterized by osteolysis. Levan polysaccharide is an active component of Erwinia herbicola, which shows potential anti-inflammatory and antioxidant properties. However, the protective effects of levan on lipopolysaccharide (LPS)-induced inflammatory and oxidative stress injury in osteoblast cells as an in vitro model of osteolysis remain largely unknown. The present study aimed to explore the functions of levan in LPS-triggered inflammation and oxidative stress in osteoblast cells (MC3T3-E1). The protective effects of levan on LPS-induced inflammatory and oxidative stress responses in MC3T3-E1 cells were assessed by quantification of superoxide dismutase (SOD) and catalase (CAT) activity as well as enzyme-linked immunosorbent assay (ELISA) for determination of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Also, the key signaling pathway of ChemR23 was determined by qPCR analysis. Results showed that levan significantly alleviated LPS-induced deactivation of SOD and CAT activity. Levan also downregulated the expression of IL-6, TNF-α, and ChemR23 at mRNA level. These findings indicated that levan may protect MC3T3-E1 cells against LPS-triggered oxidative stress, and inflammation via regulation of ChemR23. This data may provide a potential basis for further clinical investigation of levan in the prevention and treatment of LPS-triggered bone resorption and osteolysis.