Improvement of dual-leached polycaprolactone porous scaffolds by incorporating with hydroxyapatite for bone tissue regeneration

Polycaprolactone (PCL)/hydroxyapatite (HA) composite scaffolds were prepared by combining solvent casting and salt particulate leaching with a polymer leaching technique. The hydrophilicity of the dual-leached scaffold was improved by alkaline (NaOH) treatment. Well-defined interconnected pores were detected by scanning electron microscopy. The water absorption capacity of the NaOH-treated PCL/HA dual-leached scaffold increased greatly, confirming that the hydrophilicity of the scaffold was improved by NaOH treatment. The compressive modulus of the PCL/HA dual-leached scaffold was greatly increased by the addition of HA particles. An indirect evaluation of the cytotoxicity of all PCL dual-leached scaffolds with mouse fibroblastic cells (L929) and mouse calvaria-derived pre-osteoblastic cells (MC3T3-E1) indicated that the PCL dual-leached scaffolds are non-toxic to cells. The ability of the scaffolds to support mouse calvaria-derived pre-osteoblastic cell (MC3T3-E1) attachment, proliferation, differentiation, and mineralization was also evaluated. Although the viability of cells was lower on the PCL/HA dual-leached scaffold than on the tissue-culture polystyrene plates (TCPS) and on the other substrates at early time points, both the PCL and NaOH-treated PCL/HA dual-leached scaffolds supported the attachment of MC3T3-E1 at significantly higher levels than TCPS. During the proliferation period (days 1–3), all of the PCL dual-leached scaffolds were able to support the proliferation of MC3T3-E1 at higher levels than the TCPS; in addition, the cells grown on NaOH-treated PCL/HA dual-leached scaffolds proliferated more rapidly. The cells cultured on the surfaces of NaOH-treated PCL/HA dual-leached scaffolds had the highest rate of mineral deposition.     

Surface characterization and cytocompatibility of three chitosan/polycation composite membranes for guided bone regeneration

Guided bone regeneration is a promising surgical procedure for reconstructing bone defects. In this study, three chitosan/polycation composite membranes for guided bone regeneration are produced by blending chitosan with poly-L-lysine, polyethyleneimine, and poly-L-ornithine. For all composite membranes, the surface characteristics including surface topography, chemistry, and wettability are examined by atomic force microscopy, X-ray photoelectron spectroscopy, and contact angle assay. Their cytocompatibility is also evaluated with MC3T3-E1 osteoblast-like cells at cell, protein, and gene levels through cell biology assays, western blot, and RT-PCR analysis. On chitosan/poly-L-lysine composite membrane, MC3T3-E1 cells present well-developed cytoskeletal organization and significantly higher adhesion, proliferation, and differentiation than those on chitosan and the other two composite membranes. Furthermore, MC3T3-E1 cells on chitosan/poly-L-lysine membrane exhibit increased phosphorylation levels of focal adhesion kinase and extracellular signal-regulated kinase 1/2, and achieve an enhanced mRNA expression of fibronectin, Runx 2, RhoA, integrin alpha 5, and integrin beta1. From our results, we conclude that chitosan/ poly-L-lysine composite membrane possesses improved cytocompatibility with osteoblasts when compared to chitosan and holds potential for guided bone regeneration in the near future.     

Influence of multilayer rhBMP-2 DNA coating on the proliferation and differentiation of MC3T3-E1 cells seeded on roughed titanium surface

For bone morphogenetic protein (BMP) gene therapy to be a viable approach for enhancing implant osseointegration clinically, requires the development of efficient nonviral delivery vectors that can coat the implant. This study evaluated a multilayer cationic liposome-DNA complex (LDc) coating as a delivery vehicle for recombinant human BMP-2 (rhBMP-2). Multilayered coatings, comprising hyaluronic acid (HA) and LDc, were fabricated onto titanium using a layer-by-layer (LBL) assembly technique. Preosteoblastic MC3T3-E1 cells were cultured on the roughened titanium surfaces coated with multilayers of HA/LDc, or on uncoated or HA/liposome only surfaces as controls. The amount of rhBMP-2 secreted by the MC3T3-E1 cells and the effect of the various surfaces on cell viability, proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and calcium deposition were evaluated. Messenger RNA levels of OC, ALP, Runx2, and Osx were also investigated. The results demonstrated that rhBMP-2 protein secreted into culture medium at 3 days was significantly higher than control groups. MC3T3-E1 cells cultured on the HA/LDc coating displayed significantly higher ALP activity and OC secretion at 7 days and 14 days culture, respectively. MC3T3-E1 cells cultured on HA/LDc upregulated expression of the osteoblast differentiation markers, especially on days 12 for OC and on days 6 and 12 for ALP and Osx. In conclusion, MC3T3-E1 cell cultured on the multilayer HA/LDc coating surface can secret rhBMP-2 protein and the protein levels were effective in inducing early osteogenic differentiation. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2766-2774, 2012.     

Preparation and characterization of nano-hydroxyapatite/chitosan composite scaffolds

A novel nano-hydroxyapatite (HA)/chitosan composite scaffold with high porosity was developed. The nano-HA particles were made in situ through a chemical method and dispersed well on the porous scaffold. They bound to the chitosan scaffolds very well. This method prevents the migration of nano-HA particles into surrounding tissues to a certain extent. The morphologies, components, and biocompatibility of the composite scaffolds were investigated. Scanning electron microscopy, porosity measurement, thermogravimetric analysis, X-ray diffraction, X-ray photoelectron spectroscopy, and Fourier transformed infrared spectroscopy were used to analyze the physical and chemical properties of the composite scaffolds. The biocompatibility was assessed by examining the proliferation and morphology of MC 3T3-E1 cells seeded on the scaffolds. The composite scaffolds showed better biocompatibility than pure chitosan scaffolds. The results suggest that the newly developed nano-HA/chitosan composite scaffolds may serve as a good three-dimensional substrate for cell attachment and migration in bone tissue engineering.     

Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering

Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.     

In vitro response of osteoblast-like and odontoblast-like cells to unsubstituted and substituted apatites

Different types of calcium phosphate compounds [calcium-deficient apatite (CDA); beta-tricalcium phosphate (beta-TCP); biphasic calcium phosphate (BCP)] are commercially available for medical and dental applications as bone substitute materials. Most of the reported in vitro studies on cell-material interactions have used osteoblast-like cells. The purpose of this study was to investigate the in vitro response of osteoblast-like (MC3T3-E1) and odontoblast-like (MDPC23) cells on unsubstituted (HA) and substituted (F-substituted) apatites. MC3T3-E1 and MDPC23 were cultured in alpha-modified medium containing 10% fetal bovine serum, ascorbic acid (50 microg/mL) and beta-glycerophosphate (2 mM). The cells were seeded on pellets made from HA, and FAp (with low, medium, and high F concentrations). Cell morphology was observed after 7 and 14 days using scanning electron microscopy (SEM). Cell attachment and differentiation were determined from the DNA content, alkaline phosphatase (ALP) activity, and total collagen content. Pellet surface composition was characterized by using Fourier Transform infrared spectroscopy. MC3T3-E1 and MDPC23 cells on HA were normal in shape and in fusion but not on FAp. Results of this study showed that the pattern of cell proliferation of osteoblast-like cells was different from that of the odontoblast-like cells. This study suggests that cell morphology, fusion, and proliferation on biomaterial surfaces depend on cell type (osteoblast-like vs odontoblast-like cell) and biomaterial composition (unsubstituted vs substituted F-apatites).     

纳米壳聚糖对MC3T3-E1成骨细胞生长的影响

背景：已有体内急性毒理实验证实,壳聚糖纳米微囊的半数致死量高于2 000 mg/kg,但其具体致病机制目前尚不明确。 目的：分析纳米壳聚糖作为骨替代材料对MC3T3-E1成骨细胞生长及大鼠肝、肾等器官生理功能的影响。 方法：将MC3T3-E1成骨细胞分别在含0(对照)、10 mg/L、100 mg/L、1 g/L、10 g/L纳米壳聚糖的DMEM培养液中培养,检测各组细胞A值。透射电镜观察10 g/L纳米壳聚糖溶液培养MC3T3-E1成骨细胞24 h后的细胞形态变化。采用PBS制备10 g/L纳米壳聚糖悬浮液,分别以166.67,16.67 mg/kg经腹腔注射至SD大鼠体内4周,每周3次,正常对照组注射等量生理盐水,血清生化指标分析大鼠肝、肾功能,病理切片观察组织形态学改变、炎症细胞浸润情况。 结果与结论：与对照组比较,10 mg/L、100 mg/L、1 g/L、10 g/L的纳米壳聚糖溶液均抑制MC3T3-E1细胞的生长(P < 0.05)。透射电镜见团聚的壳聚糖存在于MC3T3-E1细胞浆中,细胞表面的伪足形成,细胞膜呈波浪状起伏,细胞核变性、碎裂及固缩。与正常对照组比较,注射纳米壳聚糖悬浮液两组大鼠血尿素氮、Na+水平均有明显升高(P < 0.05),高剂量组K+水平明显降低(P < 0.01);肝脏、肾脏均出现组织细胞凋亡现象,高剂量组凋亡更加明显。表明纳米壳聚糖可导致细胞凋亡,超过一定剂量可造成肾功能受损,对机体生理功能造成影响。     

In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170-200microm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.     

新型氯化锂复合磷酸钙骨水泥的理化性能及成骨特性

BACKGROUND: Lithium chloride is a widely used inorganic ion inhibitor of glycogen synthase kinase-3β, and it can be combined with glycogen synthase kinase-3β to activate the classical Wnt/β-catenin pathway, thereby promoting human bone marrow mesenchymal stem cells and osteoblasts proliferation and accelerating bone repair. OBJECTIVE: To observe the physicochemical properties of novel lithium chloride/calcium phosphate cement, and to explore its osteoinductive biological property. METHODS: Calcium phosphate cement served as control group, and lithium chloride/calcium phosphate cement containing different lithium content as experimental groups. The setting time and compressive strength of bone cement in each group were detected, and the microstructure of the material surface observed under scanning electron microscopy. Bone cement and MC3T3-E1 cells were co-cultured in vitro, and the growth and adhesion morphology of MC3T3-E1 cells on the surface of bone cement were observed under the scanning electron microscope. Effect of bone cement extracts on cell proliferation was determined through MTT assay, and alkaline phosphatase kit used for determining alkaline phosphatase activity. RESULTS AND CONCLUSION: Lithium chloride/calcium phosphate cement had the same physicochemical properties to the calcium phosphate cement. Initial and final setting time, compressive strength and morphology of bone cement had no significant differences among groups. MC3T3-E1 cells grew and adhered well on the material surface. Results of MTT assay showed that compared with the calcium phosphate cement, the lithium chloride/calcium phosphate cement was better to improve osteoblast proliferation in vitro. In addition, the alkaline phosphatase activity in MC3T3-E1 cells was higher in experimental groups than the control group. These findings indicate that lithium chloride/calcium phosphate cement can maintain good physicochemical properties, and release lithium ions to promote bone formation.       

The behavior of MC3T3-E1 cells on chitosan/poly-L-lysine composite films: effect of nanotopography, surface chemistry, and wettability

In the present work, a series of composite films were produced from chitosan/poly-L-lysine blend solutions. The surface topography, chemistry, and wettability of composite films were characterized by atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and contact angle assay, respectively. For all composite films, blending with poly-L-lysine induced changes in surface chemistry and wettability. Interestingly, it was also found that increasing poly-L-lysine weight fraction in blend solutions could result in different nanoscaled surface topographic features, which displayed particle-, granule-, or fiber-dominant morphologies. MC3T3-E1 osteoblast-like cells were cultured on all composite films to evaluate the effects of surface nanotopography, chemistry, and wettability on cell behavior. The observations indicated that MC3T3-E1 cell behavior was affected by surface topography, chemistry, and wettability simultaneously and that cells showed strong responses to surface topography. On fiber-dominant surface, cells fully spread with obvious cytoskeleton organization and exhibited significantly higher level of adhesion and proliferation compared with particle- or granule-dominant surfaces. Furthermore, fiber-dominant surface also induced greater expression of mature osteogenic marker osteocalcin and higher mineralization based on RT-PCR and von Kossa staining. The results suggest that topographic modification of chitosan substratum at the nanoscale may be exploited in regulating cell behavior for its applications in tissue engineering.     

An in vitro study of two GAG-like marine polysaccharides incorporated into injectable hydrogels for bone and cartilage tissue engineering

Natural polysaccharides are attractive compounds with which to build scaffolds for bone and cartilage tissue engineering. Here we tested two non-standard ones, HE800 and GY785, for the two-dimensional (2-D) and three-dimensional (3-D) culture of osteoblasts (MC3T3-E1) and chondrocytes (C28/I2). These two glycosaminoglycan-like marine exopolysaccharides were incorporated into an injectable silylated hydroxypropylmethylcellulose-based hydrogel (Si-HPMC) that has already shown its suitability for bone and cartilage tissue engineering. Results showed that, similarly to hyaluronic acid (HA) (the control), HE800 and GY785 significantly improved the mechanical properties of the Si-HPMC hydrogel and induced the attachment of MC3T3-E1 and C28/I2 cells when these were cultured on top of the scaffolds. Si-HPMC hydrogel containing 0.67% HE800 exhibited the highest compressive modulus (11kPa) and allowed the best cell dispersion, especially of MC3T3-E1 cells. However, these cells did not survive when cultured in 3-D within hydrogels containing HE800, in contrast to C28/I2 cells. The latter proliferated in the microenvironment or concentrically depending on the nature of the hydrogel. Among all the constructs tested the Si-HPMC hydrogels containing 0.34% HE800 or 0.67% GY785 or 0.67% HA presented the most interesting features for cartilage tissue engineering applications, since they offered the highest compressive modulus (9.5-11kPa) while supporting the proliferation of chondrocytes.     

Enhancing effect of poly(L-lactide) on the differentiation of mouse osteoblast-like MC3T3-E1 cells

Poly(L-lactide) (PLLA) has bioabsorbability and biocompatibility, and it is used as biodegradable screws, pins and plates for internal bone fixation. The purpose of this study was to clarify the effects of low molecular weight (Mw) PLLA on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. MC3T3-E1 cells were cultured with the concentration of 5-50 microg/ml of PLLA with weight average Mw of 5000 (PLLA-5k) and 10,000 (PLLA-10k) for 2 weeks using the micromass culture. Both PLLAs did not affect the proliferation of MC3T3-E1 cells. However, the calcifications of MC3T3-E1 cells were stimulated with increasing the concentration of the PLLAs. Then PLLA-5k increased the calcification of MC3T3-E1 cells more than PLLA-10k. Additionally, both PLLAs increased the alkaline phosphatase (ALP) activity and calcium content of MC3T3-E1 cells up to the similar level to the calcification. These results indicated that low Mw PLLA enhanced the differentiation of MC3T3-E1 cells with no effect on the proliferation. Moreover, it was suggested that the increase of the ALP activity was a key step to stimulate the calcification of MC3T3-E1 cells. The osteoconductivity of implanted PLLA would be based on the enhancing effect of low Mw PLLA on the differentiation of the osteoblasts.     

牵张应力介导细胞成骨分化及相关基因的表达

背景：ERK1/2信号通路和核因子κB信号通路是否参与了牵张应力作用下MC3T3-E1细胞成骨分化及相关基因表达的调控,尚不清楚。 目的：观察机械牵张应力对作用下ERK1/2和核因子κB通路对成骨细胞碱性磷酸酶、Ⅰ型胶原、骨钙蛋白、白细胞介素6表达的影响,探讨ERK1/2与核因子κB信号通路对成骨细胞分化的调控作用。 方法：体外培养的MC3T3-E1细胞,以ERK1/2通路特异性抑制剂PD098059及核因子κB通路抑制剂PDTC分别处理30 min后加载12%的拉伸应变率24 h,以正常细胞及单纯加载12%牵张应力24 h为对照。采用ELISA及Real-time PCR方法检测细胞加载前后碱性磷酸酶活性、Ⅰ型胶原、骨钙蛋白及白细胞介素6 mRNA的表达。 结果与结论：在12%牵张应力作用下,MC3T3-E1细胞碱性磷酸酶、Ⅰ型胶原、白细胞介素6的表达受ERK1/2信号通路的调控,而骨钙蛋白基因表达的变化不受ERK1/2通路的影响。核因子κB信号通路抑制剂PDTC可显著抑制机械牵应张力作用下MC3T3-E1细胞碱性磷酸酶活性的降低,同时抑制白细胞介素6基因的表达,而Ⅰ型胶原、骨钙蛋白基因表达的变化不受核因子κB信号通路的影响。结果表明牵张应力可以通过ERK1/2和核因子κB通路影响MC3T3-E1细胞的成骨分化及相关基因表达。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Effect of borate glass composition on its conversion to hydroxyapatite and on the proliferation of MC3T3-E1 cells

Glasses containing varying amounts of B(2)O(3) were prepared by partially or fully replacing the SiO(2) in silicate 45S5 bioactive glass with B(2)O(3). The effects of the B(2)O(3) content of the glass on its conversion to hydroxyapatite (HA) and on the proliferation of MC3T3-E1 cells were investigated in vitro. Conversion of the glasses to HA in dilute (20 mM) K(2)HPO(4) solution was monitored using weight loss and pH measurements. Proliferation of MC3T3-E1 cells was determined qualitatively by assay of cell density at the glass interface after incubation for 1 day and 3 days, and quantitatively by fluorescent measurements of total DNA in cultures incubated for 4 days. Higher B(2)O(3) content of the glass increased the conversion rate to HA, but also resulted in a greater inhibition of cell proliferation under static culture conditions. For a given mass of glass in the culture medium, the inhibition of cell proliferation was alleviated by using glasses with lower B(2)O(3) content, by incubating the cell cultures under dynamic rather than static conditions, or by partially converting the glass to HA prior to cell culture.     

周期性机械牵拉通过上调Runx2表达促进MC3T3-E1细胞迁移

目的 探究周期性牵拉对小鼠胚胎成骨细胞MC3T3-E1迁移功能的影响及其相关机制。方法 使用应变加载系统对体外培养的MC3T3-E1细胞施加15%幅度的牵拉,模拟细胞体内受力情况。使用划痕愈合实验检测MC3T3-E1细胞的迁移功能,使用蛋白免疫印迹法检测Runx2表达情况,使用RNA干扰技术特异性降低Runx2表达量。结果 幅度15%、频率1.25 Hz、持续24 h的周期性机械牵拉可以促进MC3T3-E1细胞的迁移,升高细胞内Runx2表达水平。在静态培养条件下,干扰Runx2能抑制MC3T3-E1细胞的迁移。干扰MC3T3-E1细胞中Runx2表达可以部分降低机械牵拉对细胞迁移的促进效应。结论 周期性牵拉可以促进MC3T3-E1细胞的迁移,在此过程中Runx2可能发挥重要作用。研究结果为寻找促进骨折愈合的创新临床治疗方法提供实验依据。     

Mechanical and in vitro biological performances of hydroxyapatite–carbon nanotube composite coatings deposited on Ti by aerosol deposition

Hydroxyapatite (HA)–carbon nanotube (CNT) composite coatings on Ti plate, produced by aerosol deposition using HA–CNT powders, were developed for biomedical applications. For the deposition process HA–CNT powder mixtures with CNT contents of 1 and 3 wt.% were used. Dense coatings with a thickness of 5 μm were fabricated, irrespective of the content of CNTs. No pores or microcracks were observed in the coatings. The coatings had good adhesion to the substrate, exhibiting a high adhesion strength, ranging from 27.3 to 29.0 MPa. Microstructural observation using field-emission gun scanning electron microscopy and transmission electron microscopy showed that CNTs with a typical tubular structure were found in the HA–CNT composite coatings. Nanoindentation tests revealed that the mechanical properties, such as the hardness and elastic modulus, were significantly improved by the addition of the CNTs to the HA coating. In addition, the proliferation and alkaline phosphatase (ALP) activity of MC3T3-E1 pre-osteoblast cells grown on the HA–CNT composite coatings were higher than those on the bare Ti and pure HA coating. The ALP activity of the composite coatings considerably improved as the CNT content increased. These results suggest that CNTs would be an effective reinforcing agent to enhance both the mechanical and biological performances of HA coatings.     

Cellular compatibility of bone-like apatite containing silicon species

A calcium carbonate/siloxane-containing poly-(lactic acid) composite (Si-CCPC) was prepared by a sol-gel method. Si-CCPC was immersed in simulated body fluid for 3 days, resulting in the formation of bone-like hydroxyapatite layer containing silicon species (b-HA(Si)) on Si-CCPC. The b-HA(Si) was estimated in the cellular compatibility by culture tests using osteoblast-like cells (MC3T3-E1 cells), human mesenchymal stem cells (MSCs) and human osteoblasts (HOBs). Cellular number of MC3T3-E1 cells and alkaline phosphatase activity of MSCs on the b-HA(Si) increased significantly more in comparison with that on the conventional b-HA (without silicon species). Cellular spreading of MC3T3-E1 cells after 1 day of culturing was enhanced on the layer with and without silicon species. The b-HA(Si) was expected to enhance cellular proliferation and osteogenic differentiation, and the b-HA layer was believed to play the important role for cellular spreading. After 14 and 21 days of HOBs-culturing, bone nodules formation was observed on the b-HA(Si). The b-HA(Si) has great potential in use as scaffolds for bone tissue engineering.     

Icariin stimulates MC3T3-E1 cell proliferation and differentiation through up-regulation of bone morphogenetic protein-2

Previous studies suggest that icariin has anabolic effects on bone, but the mechanisms are unknown. We aimed to investigate the osteogenic effects of icariin in an undifferentiated osteoblast cell line by detecting cell morphology, viability, cell cycling and bone morphogenetic protein-2 (BMP-2) expression. We treated pre-osteoblastic MC3T3-E1 cells with different concentrations of icariin [0 (as a control), 10, 20 and 40 ng/ml] for 48, 72 and 96 h. Cell morphology, viability and the cell cycle were examined and measured using microscopy, the MTT assay or flow cytometry, respectively. BMP-2-positive cells and BMP-2 protein expression levels in icariin-treated MC3T3-E1 cells were examined using immunohistochemistry staining with fluorescence optical density analysis and Western blotting. MC3T3-E1 cells showed typical characteristics of osteoblasts in response to treatment with icariin. Cells treated with all concentrations of icariin had increased percentages of S-phase cells and decreased percentages of G1-phase cells, especially in the 10 and 20 ng/ml icariin groups. The number of BMP-2-positive cells and BMP-2 protein expression levels in the 10 and 20 ng/ml icariin treatment groups were greater compared to the 0 and 40 ng/ml groups. Treatment of icariin promotes osteoblast MC3T3-E1 proliferation and differentiation in vitro, potentially owing to its role in increasing BMP-2 protein expression. Icariin potentially can be used as a drug in clinical settings to treat osteoporosis.     

In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres

Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-l-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications.     

小分子肽对成骨前体细胞MC3T3-E1骨保护素和核转录因子κB受体活化因子配体表达的影响

背景：体内实验显示,小分子肽能明显增加去卵巢大鼠的骨钙含量,使其骨密度增加,能很好地预防骨质疏松。同时体外实验显示,小分子肽能促进小鼠成骨细胞和成骨前体细胞MC3T3-E1增殖、分化、矿化,并且可能是通过抑制核转录因子 p50和p65的表达来起作用。而小分子肽对骨保护素/核转录因子κB受体活化因子配体的影响尚不明确。 目的：观察小分子肽对MC3T3-E1在增殖、分化、矿化过程中骨保护素和RANKL表达的影响。 方法：以体积分数10%胎牛血清的DMEM培养液为空白对照组,50,100 mg/L质量浓度小分子肽作用小鼠成骨前体细胞MC3T3-E1,分别于作用3,6,12,18,24,30 d后,收集细胞提取蛋白,Western Blot检测骨保护素和核转录因子κB受体活化因子配体蛋白的表达。 结果与结论：50,100 mg/L小分子肽作用MC3T3-E1后能明显促进作用骨保护素的表达(P < 0.01),而对核转录因子κB受体活化因子配体无明显影响。小分子肽作用后MC3T3-E1中骨保护素/核转录因子κB受体活化因子配体的比值要明显高于空白对照组(P < 0.01)。因此,认为小分子肽可以通过增加骨保护素的表达来影响骨保护素/核转录因子κB受体活化因子配体系统,间接地抑制破骨细胞的数量和功能。