Mandibular repair in rats with premineralized silk scaffolds and BMP-2-modified bMSCs

Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue-engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ-transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks after post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue-engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue-engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.     

Effect of BMP-9 and its derived peptide on the differentiation of human white preadipocytes

Several studies have shown that bone morphogenetic proteins (BMPs) can influence adipogenic and osteogenic cell lineages. We have shown that a peptide derived from BMP-9 (pBMP-9) at 400 ng/ml inhibits the proliferation of preosteoblasts and induces differentiation. We have now determined the effects of pBMP-9 (400 ng/ml) and equimolar concentrations of BMP-2 (100 ng/ml), BMP-9 (84.6 ng/ml) and pBMP-9 (9.04 ng/ml) on human white preadipocytes (HWP). pBMP-9 dose dependently reduced the proliferation of HWP without affecting the number of apoptotic cells. Incubation of the cells for 1 h with BMP-2, BMP-9 or pBMP-9 activated the Smad1/5/8 pathway, while incubation for 7 days in adipocyte differentiation (AD) serum-free medium containing ciglitazone and equimolar concentrations of BMP-2, BMP-9 or pBMP-9 enhanced the levels of mRNA of the adipogenic markers aP2 and adipoQ and increased the number of lipid vesicles. Thus, pBMP-9, like BMP-9, can increase the AD of HWP in AD serum-free medium.     

Bone Morphogenetic Protein-9 Induces Osteogenic Differentiation of Rat Dental Follicle Stem Cells in P38 and ERK1/2 MAPK Dependent Manner

Dental follicle stem cells are a group of cells possessing osteogenic, adipogenetic and neurogenic differentiations, but the specific mechanism underlying the multilineage differentiation remains still unclear. Great attention has been paid to bone morphogenetic protein-9 (BMP-9) due to its potent osteogenic activity. In the present study, rat dental follicle stem cells were isolated and purified, and cells of passage 3 underwent adenovirus mediated BMP-9 gene transfection to prepare dental follicle stem cells with stable BMP-9 expression. Detection of alkaline phosphatase (ALP) and calcium deposition showed dental follicle stem cells transfected with BMP-9 gene could significantly promote the osteogenesis. In addition, SB203580 and PD98059 were employed to block the p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2), respectively. Detection of ALP and calcium deposition revealed the BMP-9 induced osteogenic differentiation of dental follicle stem cells depended on MAPK signaling pathway.     

骨形态发生蛋白2基因转染犬牙髓细胞

背景：研究发现骨形态发生蛋白2具有诱导间充质细胞向成骨细胞表型分化以及诱导新骨及修复性牙本质形成的能力。 目的：通过对细胞超微结构的分析,探讨骨形态发生蛋白2基因转染的犬牙髓细胞,向成牙本质细胞转化的过程。 方法：将培养的性状稳定的第4代犬牙髓细胞分为3组：基因转染组转染pEGFP-N1-BMP-2基因,空白载体转染组转染EGFP-N1空白荧光载体,未转染组不转染。 结果与结论：成功构建pEGFP-N1-BMP-2真核表达质粒,并成功将其转染犬牙髓细胞, pEGFP-N1-BMP-2质粒转染促进犬牙髓细胞分泌骨形态发生蛋白2。pEGFP-N1-BMP-2质粒转染有促进牙髓细胞碱性磷酸酶活性的作用。透射电镜观察结果显示：转染后的犬牙髓细胞具有成牙本质细胞的形态特点。说明pEGFP-N1-BMP-2 真核表达质粒转染后的牙髓细胞,具有成牙本质细胞的特征。     

重组pcDNA3.1-hBMP-2转染兔脂肪干细胞体外诱导的成骨分化

背景：骨形态发生蛋白2具有很强的诱导干细胞成骨活性。 目的：构建人骨形态发生蛋白2基因真核表达载体,探讨其转染脂肪干细胞后的成骨效果。 方法：通过噬菌斑原位杂交筛选人混合细胞cDNA文库获得人骨形态发生蛋白2基因,与真核表达载体pcDNA3.1-连接,构建重组质粒pcDNA3.1-hBMP-2。利用脂质体Lipofectamine™ 2000分别介导人骨形态发生蛋白2 基因、EGFP基因转染第4代脂肪干细胞,并经G418进行筛选。 结果与结论：酶切鉴定及DNA测序结果证实重组质粒pcDNA3.1-hBMP-2构建成功。经计算脂质体介导的脂肪干细胞瞬时转染率为(18.0±0.42)%,并经过G418筛选后获得了稳定转染的细胞。细胞生长曲线表明转染后对脂肪干细胞生长、增殖无明显影响。ELISA检测发现人骨形态发生蛋白2组的人骨形态发生蛋白2因子表达量均高于EGFP组及未转染组,且能够稳定表达。经人骨形态发生蛋白2基因转染的脂肪干细胞的Ⅰ型胶原含量、碱性磷酸酶活性以及钙结节数目均比EGFP组及未转染组有明显升高。     

Angiomyogenesis using liposome based vascular endothelial growth factor-165 transfection with skeletal myoblast for cardiac repair

We aim to investigate the feasibility and efficacy of cholesterol (Chol)+DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (hVEGF(165)) gene transfer into human skeletal myoblasts (hSkM) for cardiac repair. The feasibility and efficacy of CD liposome for gene transfer with hSkM was characterized using plasmid carrying enhanced green fluorescent protein (pEGFP). Based on the optimized transfection procedure, hSkM were transfected with CD lipoplexes carrying plasmid-hVEGF(165) (CD-phVEGF(165)). The genetically modified hSkM were transplanted into rat heart model of acute myocardial infarction. Flow cytometry revealed that about 7.99% hSkM could be transfected with pEGFP. Based on the optimized transfection condition, transfected hSkM expressed hVEGF(165) up to day-18 (1.7+/-0.1ng/ml) with peak at day-2 (13.1+/-0.52ng/ml) with >85% cell viability. Animal studies revealed that reduced apoptosis, improved angiogenesis with blood flow in group-3 animal's heart were achieved as compared to group-1 and 2. Ejection fraction was best recovered in group-3 animals. The study demonstrates that though gene transfection efficiency using CD liposome mediated hVEGF(165) gene transfer with hSkM was low; hVEGF(165) gene expression efficiency was sufficient to induce neovascularization, improve blood flow and injured heart function.     

Effect of Low-Frequency Pulsatile Flow on Expression of Osteoblastic Genes by Bone Marrow Stromal Cells

Perfusion culture of osteoprogenitor cells is a promising means to form a bone-like extracellular matrix for tissue engineering applications, but the mechanism by which hydrodynamic shear stimulates expression of bone extracellular matrix (ECM) proteins is not understood. Osteoblasts are mechanosensitive and respond differently to steady and pulsatile flow. Therefore, to probe the effect of flow, bone marrow stromal cells (BMSCs)—cultured under osteogenic conditions—were exposed to steady or pulsatile flow at frequencies of 0.015, 0.044, or 0.074 Hz. Following 24 h of stimulus, cells were cultured statically for an additional 13 days and then analyzed for the expression of bone ECM proteins collagen 1α1 (Col1α1), osteopontin, osteocalcin (OC), and bone sialoprotein (BSP). All mRNA levels were elevated by flow, but OC and BSP were enhanced modestly with pulsatile flow. To determine if these effects were related to gene induction during flow, BMSCs were again exposed to steady or pulsatile flow for 24 h, but then analyzed immediately for expression of growth and differentiation factors bone morphogenetic proteins (BMP)-2, -4, and -7, transforming growth factor (TGF)-β₁, and vascular endothelial growth factor-A. All growth and differentiation factors were significantly elevated by flow, except BMP-4 which was suppressed. In addition, expression of BMP-2 and -7 were enhanced and TGF-β₁ suppressed by pulsatile flow relative to steady flow. These results demonstrate that pulsatile flow modulates expression of BMP-2, -7, and TGF-β₁ and suggest that enhanced expression of bone ECM proteins by pulsatile flow may be mediated through the induction of BMP-2 and -7.     

Vascularization and bone regeneration in a critical sized defect using 2-N,6-O-sulfated chitosan nanoparticles incorporating BMP-2

An ideal bone tissue engineering graft should have both excellent pro-osteogenesis and pro-angiogenesis to rapidly realize the bone regeneration in vivo. To meet this goal, 2-N,6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was successfully developed and showed a dose-dependent enhancement on angiogenesis in vitro. For the repair of a critical sized defect in rabbit radius, we developed BMP-2 loaded S-NP (BMP-2/S-NP) with protein loading efficiency of 1.4 ± 0.2% and fabricated a gelatin sponge (G) based implant loaded with BMP-2/S-NP (BMP-2/S-NP/G). This implant exerted a delivery of BMP-2 with an initial burst release of 15.3 ± 4.1% in first 24 h and a gradual release for 21 days to 77.8 ± 3.6%. The in vitro ALP assay revealed that the activity of released BMP-2 from BMP-2/S-NP/G was maintained after 3-d and 7-d delivery and further enhanced after 14-d delivery compared with the original BMP-2. Furthermore, the in vivo effects of BMP-2/S-NP/G on the bone regeneration and vessel formation in the critical sized defect (18 mm) of rabbit radius were investigated by synchrotron radiation-based micro-computed tomography (SRμCT) imaging, three dimensional micro-computed tomographic (μCT) imaging, histological analysis, immunohistochemistry and biomechanical measurement. Based on the results, both peripheral vessel and new vessel formation were significantly increased by the BMP-2/S-NP/G treatment, along with the bridged defects at as early as 2 weeks, the healed defects at 8 weeks and the reunion of bone marrow cavity at 12 weeks. The results indicated that both controlled release of active BMP-2 and favorable vascularization at the defect site contributed by BMP-2/S-NP/G played a crucial role in accelerating and promoting bone augmentation. This study suggests that BMP-2/S-NP/G demonstrates promise for vascularization and bone regeneration in clinical case of large defect.     

The evaluation of ectopic bone formation induced by delivery systems for bone morphogenetic protein-9 or its derived peptide

We have earlier shown that a peptide derived from the bone morphogenetic protein-9 (pBMP-9) stimulates mouse preosteoblasts MC3T3-E1 differentiation in vitro. Here, we evaluated the effects of two delivery systems (DSs) for pBMP-9, one based on collagen and the other on chitosan. The release kinetics of BMP-9 (used as control) and pBMP-9 from these DSs were first determined in vitro by using enzyme-linked immunosorbent assay and high performance liquid chromatography assays, respectively. Micro-computerized tomography and histological analysis were then performed to study in vivo the ectopic ossification induced by both DSs containing these molecules in C57BL/6 mouse quadriceps. We found that collagen DS released in vitro about 35% of its BMP-9 within 1?h, whereas chitosan DS released 80%. The pBMP-9 was released from both DSs more slowly for up to 10 days. These release kinetics seemed to fit the Korsmeyer-Peppas model. Only chitosan DS containing BMP-9 induced strong bone formation in all mice quadriceps within 24 days. All mice quadriceps treated by pBMP-9 trapped in this DS also favored bone structures that started to mineralize. However, pBMP-9 in collagen DS failed to promote ectopic ossification within 24 days in vivo. This study highlights the importance to optimize carrier, thus improving the efficiency of pBMP-9 in vivo.     

STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation

Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium.     

骨形态发生蛋白2基因转染人羊水干细胞复合β-磷酸三钙支架促进骨再生

目的研究重组腺病毒介导的骨形态发生蛋白2（BMP-2）基因转染人羊水干细胞（hAFSC）的体外成骨分化,评价基因修饰的hAFSC负载于β-磷酸三钙（β-TCP）多孔支架后体内成骨能力。方法收集人工破膜后的中段羊水原代培养hAFSC,免疫磁珠分选CDl17／c-kit阳性的hAFSC,分别转染腺病毒介导的BMP-2或增强型绿色荧光蛋白（EGFP）基因,转染组转染BMP-2和EGFP基因、对照组转染无BMP-2编码序列的EGFP基因,空白组不做病毒转染。48h后荧光显微镜下观察细胞荧光表达及生长状况,应用流式细胞仪检测转染率。转染第7、14、21、28天采用ELISA检测各组hAFSC培养液中的BMP．2分泌量以确认转染后目的蛋白表达效果。各组hAFSC成骨诱导14d后进行碱性磷酸酶（ALP）染色,28d后进行茜素红染色评价成骨能力;成骨诱导3、7、14d后定量检测各组细胞ALP活性。20只8周龄裸小鼠随机分为Adv-hBMP-2-hAFSC-β-TCP组、Adv-EGFP-hAFSC-β-TCP组、hAFSC-β-TCP组和β-TCP组,每组5只,将细胞载体复合物植入各组裸小鼠股骨,8周后观察异位成骨组织学情况。结果hAFSC生长良好,Adv-hBMP-2或Adv-EGFP基因转染48h后荧光显微镜下见细胞贴壁良好,发出强绿色荧光,无死细胞产生,转染阳性率达（89．00±4．25）％。转染Adv-hBMP-2组hAFSC培养液上清中BMP-2第7、14、21、28天分别为（3．405±0．229）μg／L、（4．575±0．179）μg／L、（3．910±0．175）μg／L、（2．694±0．205）μg/L,第14天蛋白BMP-2分泌已达到高峰且第28天仍有蛋白表达,各时间点均明显高于对照组与空白组（均P〈0．05）。hAFSC成骨诱导14d后细胞相连成片,与对照组及空白组比较,ALP染色转染组染色阳性面积更大、阳性细胞数量更多。成骨诱导28d后茜素红染色见转染组细胞多中心聚集,伴大量红色钙结节形成,结节数量与大小明显优于对照组与空白组。成骨诱?     

Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration

There are several gene therapy approaches to tissue regeneration. Although usually efficient, virusbased approaches may elicit an immune response against the viral proteins. An alternative approach, nonviral transfer, is safer, and can be controlled and reproduced. We hypothesized that in vivo bone formation could be achieved using human mesenchymal stem cells (hMSCs) nonvirally transfected with the human bone morphogenetic protein-2 (hBMP-2) or -9 (hBMP-9) gene. Human MSCs were transfected using nucleofection, a unique electropermeabilization-based technique. Postnucleofection, cell viability was 53.6 +/- 2.5% and gene delivery efficiency was 51% to 88% (mean 68.2 +/- 4.1%), as demonstrated by flow cytometry in enhanced green fluorescent protein (EGFP)-nucleofected hMSCs. Transgene expression lasted longer than 14 days and was very low 21 days postnucleofection. Both hBMP-2- and hBMP-9-nucleofected hMSCs in culture demonstrated a significant increase in calcium deposition compared with EGFP-nucleofected hMSCs. Human BMP-2- and hBMP-9-nucleofected hMSCs transplanted in ectopic sites in NOD/SCID mice induced bone formation 4 weeks postinjection. We conclude that in vivo bone formation can be achieved by using nonvirally nucleofected hMSCs. This could lead to a breakthrough in the field of regenerative medicine, in which safer, nonviral therapeutic strategies present a very attractive alternative.     

BMP7 induces the differentiation of bone marrow-derived mesenchymal cells into chondrocytes

Injured articular cartilage has a poor capacity for spontaneous healing. So far, a satisfactory solution to repair the injured cartilage has not been found, but transgenic therapy might be a promising treatment. This study aims to evaluate the potential of transfecting bone morphogenetic protein-7 (BMP-7), a secretory protein, into bone marrow-derived mesenchymal stem cells (BMSCs), in inducing the differentiation of bone marrow stromal cells into chondrocytes in vitro. The phenotypes of the cells were observed by alcian blue staining and H&E staining with an inverted microscope. The glycosaminoglycan (GAG) content of BMSCs transfected with pcDNA3.1-BMP7 or induced by inducing medium was examined after 7, 14, or 21 days of incubation. A standard curve as reference for BMSCs’ GAG content was plotted using galacturonic acid. The content of type II collagen in culture medium was detected by ELISA. Our results demonstrated that BMP7-transfected BMSCs or BMSCs incubated with inducing medium possess the ability to differentiate into chondrocytes. BMP7-induced BMSCs secrete type II collagen and GAG. There was no significant difference between BMP7-induced BMSCs in their secreted protein content when compared with the positive control group (TGF-β1 and dexamethasone) (P > 0.05), but there was significant difference in the secreted protein profile when compared with the negative control group (P < 0.05).     

人瘦素基因过表达增强大鼠骨髓基质细胞成骨能力

目的探讨人瘦素(h LEP)基因修饰对大鼠骨髓基质细胞(r BMSCs)成骨能力的影响。方法以转染h LEP腺病毒载体(Ad5-h LEP-EGFP)的r BMSCs为实验组,空载体(Ad5-EGFP)转染的r BMSCs和未转染的r BMSCs为对照组。MTT法检测细胞增殖情况,实时荧光定量PCR法检测Ⅰ型胶原(Col-Ⅰ)和碱性磷酸酶(ALP)mRNA的表达,茜素红染色检测矿化结节形成能力。同时,将各组r BMSCs与β磷酸三钙(β-TCP)复合后移植于裸鼠皮下培养8周,观察新骨形成情况。结果 Ad5-h LEP-EGFP能有效转染r BMSCs,转染后细胞增殖活性无明显改变,但Col-Ⅰ和ALP mRNA的表达较对照组有明显提高(P0.05),矿化结节形成能力也更强(P0.05)。而且,h LEP修饰后的r BMSCs能与β-TCP结合形成组织工程化复合物,并能在裸鼠体内形成更多的类骨样组织。结论 h LEP修饰的r BMSCs可增强细胞成骨能力,有望应用于骨及牙周组织再生研究。     

Efficient transfection method using deacylated polyethylenimine-coated magnetic nanoparticles

Low efficiencies of nonviral gene vectors, such as transfection reagent, limit their utility in gene therapy. To overcome this disadvantage, we report on the preparation and properties of magnetic nanoparticles [diameter (d) = 121.32 ± 27.36 nm] positively charged by cationic polymer deacylated polyethylenimine (PEI max), which boosts gene delivery efficiency compare with polyethylenimine (PEI), and their use for the forced expression of plasmid delivery by application of a magnetic field. Magnetic nanoparticles were coated with PEI max, which enabled their electrostatic interaction with negatively charged molecules such as plasmid. We successfully transfected 81.1 ± 4.0% of the cells using PEI max-coated magnetic nanoparticles (PEI max-nanoparticles). Along with their superior properties as a DNA delivery vehicle, PEI max-nanoparticles offer to deliver various DNA formulations in addition to traditional methods. Furthermore, efficiency of the gene transfer was not inhibited in the presence of serum in the cells. PEI max-nanoparticles may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.     

Short-term effects of adhesion peptides on the responses of preosteoblasts to pBMP-9

Adhesion peptides are currently used to enhance the interactions of osteoblasts with biomaterials. However, little is known about the effects of adhesion peptides on cell responses to growth factors, especially the bone morphogenetic proteins (BMPs). We used adhesion peptides Ac-CGGNGERPRGDTYRAY-NH(2) (pRGD), derived from bone sialoprotein, and Ac-CGGDGEA-NH(2) (pDGEA), derived from collagen, which interact with alpha(v)beta(3) and alpha(2)beta(1) integrins, respectively. We analyzed the effects of pRGD- and pDGEA-coated polystyrene (PS) on the responses of murine MC3T3-E1 preosteoblasts to a peptide derived from human BMP-9 (pBMP-9) in serum-free medium. After 1h, pRGD favoured interactions with alpha(v) while pDGEA bound beta(1) integrin subunits. Adding pBMP-9 (400 ng/mL) increased the amount of alpha(v) integrin subunits in cell membranes on pRGD-coated PS, but had no effect on beta(1) integrin subunits. Only on this substratum, collagen type I mRNA was enhanced and the addition of pBMP-9 promoted the early cell differentiation, increasing their alkaline phosphatase (ALP) activity within 24 h. These cells also organized beta(1) integrin subunits at their focal adhesion points. Inhibiting alpha(2)beta(1) integrins by pDGEA pre-treatment decreased this ALP activity. It is therefore important to understand the impact of adhesion peptides on the early cell responses to growth factors in order to improve biomimetic materials.