Evaluation of the cytocompatibility of butanediamine- and RGDS-grafted poly(dl-lactic acid)

Butanediamine-grafted polylactic acid (BDPLA) is a well-established poly(dl-lactic acid) (PDLLA) based biomaterial with pendant carboxyl groups and amino groups. It has been proved out that BDPLA possesses highly improved hydrophilicity and reduced acidity of degrading products compared with PDLLA. RGDS-PLA is a BDPLA-based biomimetic material obtained by covalently incorporating RGDS adhesion peptide. The objective of this study was to evaluate the cytocompatibility of BDPLA and RGDS-PLA with PDLLA as control. Rat calvarial osteoblasts (ROBs) were seeded on various polymer films, and the morphology, adhesion and spreading, and viability and proliferation of ROBs were assessed by scanning electronic microscopy (SEM), micropipette aspiration system and methylthiazolyl tetrazolium assay (MTT assay), respectively. ALP activity and extracellular calcium production were exploited to characterize the differentiation of ROBs on various polymer films. The affecting mechanisms of BDPLA and RGDS-PLA on the cytocompatibility were extensively explained. The results revealed that the introduction of butanediamine to PDLLA is obviously beneficial to the adhesion, spreading, growth and differentiation of ROB, and the introduction of RGDS adhesive peptide can further improve the cytocompatibility of PDLLA. These results suggest that, in terms of cytocompatibility, BDPLA and RGDS-PLA are promising biomaterials for tissue engineering and other biomedical applications, and RGDS-PLA is a better one.     

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.     

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.     

物理老化对聚乳酸玻璃化转变行为的影响

为考察不同链结构的聚乳酸在物理老化过程中聚集态结构的变化 ,分别在 0℃、2 5℃、37℃对聚 (D ,L 乳酸 ) (PDLLA)、非晶态的低左旋度聚 (L 丙交酯 ) (l PLLA)和左旋聚 (L 丙交酯 ) (PLLA)进行长达 6个月的物理老化 ,用温度调制式差示扫描量热法 (MDSC)研究了它们在物理老化过程中的玻璃化转变行为的变化 .结果表明 ,PDLLA和l PLLA对物理老化十分敏感 ,在一定老化温度下 ,它们的玻璃化转变温度 (Tg)随老化时间延长向高温移动 ,老化过程中产生的非可逆热焓 (ΔHnon)逐渐增加 ,这种趋势在老化 1w内变化明显 ,随后趋缓 ;而老化温度越高 ,Tg 升高和ΔHnon增加的速率都越快 .不同温度下老化 ,PDLLA和l PLLA的ΔHnon 与老化时间的对数都有较好的线性关系 ,ΔHnon的变化速率对温度的倒数作图 ,l PLLA的斜率绝对值比PDLLA的略大 ,说明有较高立构规整性的分子链形成物理缠结需要较高的活化能 .PLLA由于结晶度高而非晶相含量较低 ,未观察到明显的玻璃化转变及其在物理老化过程中的变化 .     

Lipase-catalyzed transformation of poly(lactic acid) into cyclic oligomers

Enzymatic transformations into cyclic oligomers were carried out with the objective of developing chemical recycling of poly(lactic acid)s, such as poly(D,L-lactic acid) (PDLLA), poly(D-lactic acid) (PDLA) and poly(L-lactic acid) (PLLA), which are typical biodegradable polymers. They were degraded by lipase in an organic solvent to produce the corresponding cyclic oligomer with a molecular weight of several hundreds. PDLLA (with a Mw of 84,000) was quantitatively transformed into cyclic oligomers by lipase RM (Lipozyme RM IM) in chloroform/hexane at 60 degrees C. PLLA (with a Mw of 120,000) was transformed into cyclic oligomer by lipase CA (Novozym 435) at a higher temperature of 100 degrees C in o-xylene. The oligomer structure was identified by 1H and 13C NMR spectroscopy and MALDI-TOF (matrix assisted laser desorption/ionization-time-of-flight) mass spectrometry.     

Competition between Liquid-liquid De-mixing,Crystallization, and Glass Transition in Solutions of PLA of Different Stereochemistry and DEET

Liquid-liquid(L-L)de-mixing and vitrification of solutions of either crystallizable poly(L-lactic acid)(PLLA)or non-crystallizable poly(D/L-lactic acid)(PDLLA)with 50 m%N,N-diethyl-3-methylbenzamide(DEET)were analyzed by calorimetry and cloud-point measurements,which allows drawing conclusions about the effect of polymer stereochemistry on the phase behavior.Regardless of the PLA stereochemistry,vitrification of the solutions on fast cooling,hindering crystallization of PLLA,occurred below-20℃ and suppressed prior L-L de-mixing.The experimental results prove that crystallization in samples containing crystallizable PLLA,observed at around 55℃ on slow cooling,is not preceded by L-L de-mixing.     

Oxygen Plasma Technology-Assisted Preparation of Three-Dimensional Reduced Graphene Oxide/Polypyrrole/Strontium Composite Scaffold for Repair of Bone Defects Caused by Osteoporosis

Repairs of bone defects caused by osteoporosis have always relied on bone tissue engineering. However, the preparation of composite tissue engineering scaffolds with a three-dimensional (3D) macroporous structure poses huge challenges in achieving osteoconduction and osteoinduction for repairing bone defects caused by osteoporosis. In the current study, a three-dimensional macroporous (150–300 μm) reduced graphene oxide/polypyrrole composite scaffold modified by strontium (Sr) (3D rGO/PPY/Sr) was successfully prepared using the oxygen plasma technology-assisted method, which is simple, safe, and inexpensive. The findings of the MTT assay and AO/EB fluorescence double staining showed that 3D rGO/PPY/Sr has a good biocompatibility and effectively promoted MC3T3-E1 cell proliferation. Furthermore, the ALP assay and alizarin red staining showed that 3D rGO/PPY/Sr increased the expression levels of ALP activity and the formation of calcified nodules. The desirable biocompatibility, osteoconduction, and osteoinduction abilities, assure that the 3D macroporous rGO/PPY/Sr composite scaffold offers promising potential for use in the repair of bone defects caused by osteoporosis in bone tissue engineering.     

Amorphous calcium phosphate/poly(D,L-lactic acid) composite nanofibers: electrospinning preparation and biomineralization

Amorphous calcium phosphate (ACP) has been recognized as an attractive biomaterial due to its bioactivity and biocompatibility. Electrospinning is a simple and low-cost way to fabricate polymer fibers. In this study, ACP nanoparticles with diameters ranging from 20 to 80 nm were synthesized using a simple precipitation method. ACP nanoparticles were hybridized with poly(D,L-lactic acid) (PDLLA) to form ACP/PDLLA composite nanofibers by electrospinning, and different architectures including the nanofibrous mesh and tube consisting of ACP/PDLLA composite nanofibers were obtained and characterized. The biomineralization and cytocompatibility of as-prepared ACP/PDLLA composite nanofibers were evaluated in vitro. Osteoblast-like MG63 cells were seeded on the ACP/PDLLA composite nanofiber meshes to perform the cytocompatibility evaluation. The ACP/PDLLA composite nanofibers exhibited a fast mineralization behavior in the simulated body fluid. The attachment of MG63 cells and cytotoxicity of ACP/PDLLA composite nanofibers were also evaluated, and the experiments indicated good biocompatibility and bioactivity of ACP/PDLLA composite nanofibers.     

The chiral effects on the responses of osteoblastic cells to the polymeric substrates

In order to study the chiral effects of polylactides on responses of osteoblastic cells, poly(l-lactide) (PLLA), poly(d-lactide) (PDLA), poly(dl-lactide) (PDLLA) and the stereocomplex of PLLA and PDLLA (SC) films with different stereoforms were prepared. The surface properties of the four polylactide films were tested and the osteoblastic ROS 17/2.8 cells were cultured on the films. The protein adsorption behaviors of fibrinogen and bovine serum albumin on films were studied. The cell proliferation, total protein amount, DNA content and alkaline phosphatase activity of osteoblastic ROS 17/2.8 cells were evaluated. The results showed that the protein adsorption was dependant on the type of proteins. The observation of cell morphologies revealed that the PDLA film provide an unfavorable surface for cell attachment. The total protein amount, DNA content and ALP activity were closely related to the stereoforms of polylactide films. All the levels of total protein amount, DNA content and ALP activity of ROS 17/2.8 cells on PDLA film were decreased. The racemic stereocomplex of PLLA and PDLA showed relatively higher positive effects on both cell growth and proliferation.     

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.

     

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.     

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.     

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.     

静电纺丝法制备胆固醇-g-聚乳酸液晶/ 聚乳酸复合纳米纤维膜

以辛酸亚锡为催化剂, 胆固醇(CHOL)为共引发剂引发D,L-丙交酯开环聚合, 制备了胆固醇-g-聚(D,L-乳酸)(CHOL-g-PDLLA)低聚物, 采用偏光显微镜(POM)和差示扫描量热(DSC)方法考察了其液晶特性. 通过静电纺丝技术制备了CHOL/PDLLA和CHOL-g-PDLLA /PDLLA复合纳米纤维膜, 对其形貌、界面相容性、孔隙率、拉伸性能和细胞相容性进行了研究. 结果表明, CHOL-g-PDLLA为一种热致胆甾型液晶, 液晶温度区间为21.8~74.5 ℃; CHOL-g-PDLLA/PDLLA复合纳米纤维膜的纤维形态良好, 表面均匀光滑, 孔隙率在70%~75%之间, 且其界面相容性优于相应的CHOL/PDLLA. 随着CHOL和CHOL-g-PDLLA含量的增加, 复合纳米纤维膜的拉伸强度逐渐下降, 但CHOL-g-PDLLA/PDLLA复合纳米纤维膜的拉伸强度显著大于CHOL/PDLLA. 体外骨髓间充质干细胞培养结果显示, CHOL-g-PDLLA/PDLLA复合纳米纤维膜具有良好的细胞相容性, 且优于相应的PDLLA和CHOL/PDLLA纳米纤维膜.     

In vitro Evaluation of Osteoblast Response to Sol-Gel Derived Gelatin-Siloxane Hybrids

Cytotoxicity and cytocompatibility of porous gelatin-siloxane hybrids were evaluated due to osteoblastic cell (MC3T3-E1) proliferation, ALP activity, or their responses to the hybrids and their extracts. The hybrids were found intoxic, and appropriate incorporation of calcium ions stimulated proliferation and differentiation in vitro. Cells were seeded into the porous hybrids and the cell morphology was studied. The hybrids involving calcium ions favored osteoblast growth and differentiation.     

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.     

Amphirionin-5, a novel linear polyketide from a cultured marine dinoflagellate Amphidinium species with a potent cell proliferation-promoting activity

A novel linear polyketide, amphirionin-5 (1), has been isolated from the cultivated algal cells of the benthic dinoflagellate Amphidinium sp. (strain KCA09053), and the structure was elucidated on the basis of detailed analyses of 1D and 2D NMR data. Amphirionin-5 (1) resulted in a 282% increase in the proliferation of murine bone-marrow derived stromal ST-2 cells and 320% increase in the proliferation of murine osteoblastic MC3T3-E1 cells.