新生大鼠颅骨成骨性细胞体外生长过程研究

为了确定鼠颅骨成骨性细胞在体外的发育分化过程是否与体内成骨细胞表型相似，我们用分次酶消化法分离新生大鼠颅骨成骨性细胞，进行形态学观察、碱性磷酸酶（Ａ１Ｐ）活性表现、细胞对甲状旁腺素（ＰＴＨ）的反应和形成钙化骨基质结节等研究。结果表明，新生大鼠颅骨细胞在体外培养过程中有两个明显的生长阶段，即增生期和分化成熟期。在增生期细胞数目逐渐增多，形态不规则，胞膜上的Ａ１Ｐ活性低，对ＰＴＨ反应逐渐增强；在分化成熟期细胞增殖缓慢，呈多边形，且聚集重叠成细胞结节，出现钙化骨基质，Ａ１Ｐ活性增强，而对ＰＴＨ的反应在第９ｄ达到高峰后却逐渐减弱。我们认为鼠颅骨成骨性细胞在体外随时间进程有着不同的生长模式，逐渐发育分化为成熟的成骨细胞。

基于FoxO1通路探讨仙茅苷对H2O2诱导成骨细胞氧化损伤的保护作用及机制

目的探讨仙茅苷(Curculigoside)对H_2O_2诱导成骨细胞氧化损伤的保护作用及机制。方法采用H_2O_2(0.3 mmol·L^(-1))处理MC3T3-E1成骨细胞建立细胞氧化损伤模型,并以阳性对照药氮乙酰半胱氨酸(NAC,2.5 mmol·L^(-1))和不同浓度仙茅苷(10^(-6)、10^(-7)、10^(-8)mol·L^(-1))进行干预。采用MTT法检测成骨细胞的增殖情况;磷酸苯二钠法(p-PNPP-Na)法测定成骨细胞碱性磷酸酶(ALP)活性;茜素红染色法观察成骨细胞的骨矿化结节形成情况;ELISA法检测细胞超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力;流式细胞仪检测细胞活性氧(ROS)和线粒体膜电位;Western Blot法检测细胞叉形头转录因子1(FoxO1)和SOD-2的蛋白表达水平;免疫荧光组织化学法检测FoxO1、活化转录因子4(ATF4)、β-连环蛋白(β-catenin)在细胞核中的表达。结果与空白对照组比较,模型组成骨细胞在H_2O_2处理36~72 h后,MC3T3-E1细胞的增殖受到显著抑制(P<0.001);成骨细胞活力及ALP活性均明显降低(P<0.05),细胞形成的骨矿化结节的数量和面积明显减少;SOD活性明显降低(P<0.05),CAT活性明显增加(P<0.05);ROS水平增加,线粒体膜电位降低,总FoxO1a、SOD-2蛋白表达下调,FoxO1a在细胞核内的表达下调,在细胞质中的表达上调,在细胞核与细胞质表达的比例降低,但差异没有统计学意义(P>0.05);细胞核中FoxO1a、β-catenin、ATF4表达量升高。与模型组比较,阳性药组和不同浓度仙茅苷组的成骨细胞活力及ALP活性均明显上调(P<0.05),且细胞骨矿化结节的数量和面积明显增加;阳性药组及仙茅苷10^(-6)mol·L^(-1)组的细胞SOD活性明显增加(P<0.05),仙茅苷10^(-6)mol·L^(-1)组细胞的CAT活性明显增加(P<0.05);10^(-7)mol·L^(-1)仙茅苷组细胞的ROS水平明显降低(P<0.05);仙茅苷10^(-8)mol·L^(-1)组的线粒体膜电位明显升高(P<0.05);阳性对照组和10-6mol·L^(-1)仙茅苷组的细胞总FoxO1a蛋白表达明显上调(P<0.05);各给药组细胞的SOD-2蛋白表达均明显上调(P<0.05);10^(-6)mol·L^(-1)仙茅苷组细胞FoxO1a在细胞核与细胞质表达的比值明显升高(P<0.05);10^(-6)mol·L^(-1)仙茅苷组细胞的FoxO1a、ATF4在细胞核的表达上调,β-catenin在细胞核的表达没有明显变化。结论仙茅苷能够保护H_2O_2对MC3T3-E1成骨细胞的氧化损伤,可能与其增加FoxO1在细胞核内的表达,增强FoxO1a与ATF4相互作用,以及增强SOD及CAT活性有关。

胎儿毛乳头细胞分化为类成骨细胞的实验研究

目的研究胎儿毛乳头细胞的体外成骨能力,为骨组织工程提供一种新的种子细胞。方法Ⅰ型胶原酶消化法分离毛乳头,常规成纤维细胞培养液培养毛乳头细胞。第3代和第7代细胞经成骨诱导液诱导培养7d,以免疫荧光、VonKossa等方法鉴定毛乳头细胞及检测细胞的成骨特性。结果培养的毛乳头细胞呈聚集性生长,表达平滑肌肌动蛋白和Ⅰ型胶原。经成骨诱导后表达骨桥蛋白,有钙结节形成。结论毛乳头细胞具有成骨活性,可作为骨组织工程的种子细胞。

Study on biocompatibility of PDLLA/HA/DBM with co-cultured human osteoblasts in vitro

Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDLLA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen Ⅰ expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of biomaterial and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.

Effect of Phytoestrogen Activity on hFOB 1.19 Osteoblast Cells of Vanilla Siamensis

The unsaponifiable compounds derived from the fresh green beans of Vanilla siamens＆ Rol. ex. Dow were assayed for the first time to detect their estrogenic activity. We used a simple screening method using the yeast two hybrid system based on the binding of a ligand to estrogen receptors. Yeast cells carrying the hER （human estrogen receptor） gene, ERE （estrogen response elements） and lacZ （β-galactosidase gene） are very suitable for screening and sensitive analysis of estrogenic compound. Our results showed that V. siamensis plant extracts bind with relatively affinity to YES- hERa was 2.27-fold the relative potency ofestradiol （E2） in YES-hERa. The effects of phytoestrogen activity on the osteoblast cells were examined on the proliferation of hFOB 1.19 cells and the bone mineralization process. V. siamens＆ was a positive screening result and induced mineralization ofosteoblasts. This study indicated that V. siamensis plant extract exhibited the characteristic effects of a nature bone promoter compound as phytoestrogen.

Effects of basic fibroblast growth factor on biological characteristics of osteoblasts

Objective: To elucidate the effects of exogenous basic fibroblast growth factor ( bFGF ) on biological characteristics of rat osteoblasts cultured in vitro.Methods: The osteoblasts isolated from a Sprague-Dawley rat and cultured in vitro were treated with different concentrations of bFGF (5-50 ng/ml) respectively. At 24 hours after treatment, the proliferating cell nuclear antigen was measured with immunocytochemistry, alkaline phosphatase ( ALP) activity was determined and the expression of transforming growth factor beta 1 (TGF-β1) was detected to observe the effects of bFGF on growth and differentiation of osteoblasts. Results: bFGF ( 5-50 ng/ml ) could obviously promote the growth of osteoblasts. The intracellular expression of TGF-β, mRNA increased significantly, but the intracellular ALP content decreased.Conclusions: bFGF can obviously stimulate the proliferation of osteoblasts and promote the synthesis of TGF-β1, but cannot promote the differentiation of osteoblasts.

Unique cytological behavior of MC3T3-E1 osteoblasts on H_2O_2-modified C/C composites in vitro

MC3T3-E1 osteoblasts were cultured on H2O2- modified and unmodified carbon/carbon （H-C/C and C/C） composites for one week in order to evaluate differences in cell adhesion, spreading, and proliferation. The results indicated a certain degree of enhancement in the cell adhesion capability of osteoblasts cultured on H-C/C samples. Cellu- lar morphologies after cell adhesion were observed via scan- ning electron microscopy （SEM）, which showed that the cells adhered more closely and spread more widely on the H-C/C sample surface. However, no cell appeared in several mul- tiple and continuous types of minor pores on both the C/C and H-C/C surfaces. In addition, two unique situations were observed on the H-C/C samples： an outline change of the osteoblasts was observed when the cells spread across some minor pores, and the cells entered and adhered well in some larger pores.

Polydopamine-assisted functionalization of heparin and vancomycin onto microarc-oxidized 3D printed porous Ti6Al4V for improved hemocompatibility,osteogenic and anti-infection potencies

Enhanced antiinfection activities, improved hemocompatibility and osteo-compatibility, and reinforced osseointegration are among the most important considerations in designing multifunctional orthopedic biomaterials.Hereby, anti-infective and osteogenic multifunctional 3 D printed porous Ti6 Al4 V implant with excellent hemocompatibility was successfully designed and fabricated. In brief, osteogenic micro-arc oxidation（MAO） coatings with micro/nanoscale porous topography were generated in situ on3 D printed Ti6 Al4 V scaffolds, on which heparin and vancomycin were easily immobilized. The surface microstructure,morphology, and chemical compositions were characterized employing scanning electron microscopy（SEM）, X-ray photoelectron spectroscopy（XPS） and Fourier transform infrared spectroscopy（FTIR）. High loading capacity and sustained vancomycin release profiles were revealed using high performance liquid chromatography（HPLC）. Favorable antibacterial and antibiofilm performances against pathogenic Staphylococcus aureus（S. aureus） were validated in vitro through microbial viability assays, Live/Dead bacterial staining, and crystal violet staining. Human mesenchymal stem cells（h MSCs） were seeded on the scaffolds and their proliferation and viability were assessed using Cell Counting Kit and Live/Dead cell viability kit. Further, osteoblastic differentiation abilities were evaluated using alkaline phosphatase（ALP） activity as a hall marker. Additionally, the improved hemocompatibility of the heparinized scaffolds was confirmed by activated partial thromboplastin time（APTT）, prothrombin time（PT） and thrombin time（TT）. Overall, our results show that the surface-modified 3 D printed porous Ti6 Al4 V possesses balanced antibacterial and osteogenic functions while exhibiting extra anticlotting effects, boding well for future application in customized functional reconstruction of intricate bone defects.