Differentiation stage alters matrix control of stem cells

Cues from the material to which a cell is adherent (e.g., adhesion ligand presentation, substrate elastic modulus) clearly influence the phenotype of differentiated cells. However, it is currently unclear if stem cells respond similarly to these cues. This study examined how the overall density and nanoscale organization of a model cell adhesion ligand (arginine-glycine-aspartic acid [RGD] containing peptide) presented from hydrogels of varying stiffness regulated the proliferation of a clonally derived stem cell line (D1 cells) and preosteoblasts (MC3T3-E1). While the growth rate of MC3T3-E1 preosteoblasts was responsive to nanoscale RGD ligand organization and substrate stiffness, the D1 stem cells were less sensitive to these cues in their uncommitted state. However, once the D1 cells were differentiated towards the osteoblast lineage, they became more responsive to these signals. These results demonstrate that the cell response to material cues is dependent on the stage of cell commitment or differentiation, and these findings will likely impact the design of biomaterials for tissue regeneration.     

Heterodimeric BMP-2/7 exhibits different osteoinductive effects in human and murine cells

As robust osteoinductive cytokines, bone morphogenetic proteins (BMPs) play a significant role in bone tissue engineering. Constituted of two different polypeptides, heterodimeric BMPs are more effective than the homodimers in bone formation. While most studies focused on the murine cell lines, such as murine preosteoblasts MC3T3-E1, the role of heterodimeric BMPs in the osteogenic differentiation of human cells remains uncertain, which hinders their application to practical treatment. In this study, we compared the osteoinductive effects of BMP-2/7 heterodimer in human adipose-derived stem cells (hASCs) with their homodimers BMP-2 and BMP-7, in which MC3T3-E1 cells were utilized as a positive control. The results indicated that BMP-2/7 was not a stronger inducer during the osteogenic differentiation of hASCs as that for MC3T3-E1, and extracellular-signal-regulated kinase signaling played a role in the different effects of BMP-2/7 between hASCs and MC3T3-E1. Our study demonstrates the osteoinductive effects of heterodimeric BMP-2/7 present in a cell-specific pattern and cautions should be taken when applying heterodimeric BMP-2/7 to clinical practice.     

Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: a combined computational and experimental approach

The adhesion ligand arginine-glycine-aspartic acid (RGD) has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters key cell behaviors. Previous studies have failed, however, to conclusively decouple the effects of RGD bulk density and individual pattern parameters (i.e. RGDs/island and island distribution) on these altered cell responses. Using a nanopatterned RGD-coupled alginate hydrogel matrix, this work combines computational, statistical and experimental approaches to elucidate the effects of RGD patterns on four key cell responses. This study shows that in MC3T3 preosteoblasts focal adhesion kinase (FAK) Y397 phosphorylation, cell spreading, and osteogenic differentiation can be controlled by RGD nanopatterning, with the distribution of islands throughout the hydrogel (i.e. how closely spaced the islands are) being the most significant pattern parameter. More closely spaced islands favor FAK Y397 phosphorylation and cell spreading, while more widely spaced islands favor differentiation. Proliferation, in contrast, is primarily a function of RGD bulk density. Nanopatterning of cell adhesion ligands has tremendous potential as a simple tool to gain significant control over multiple cell behaviors in engineered extracellular matrix (ECM).     

Ultrahigh molecular weight polyethylene wear debris inhibits osteoprogenitor proliferation and differentiation in vitro

Polyethylene wear debris induces progressive osteolysis by increasing bone degradation and suppressing bone formation. Polyethylene particles inhibit the function of mature osteoblasts, but whether polyethylene particles also interfere with the proliferation and differentiation of osteoprogenitor cells is unknown. In this study, we investigated the effects of ultrahigh molecular weight polyethylene (UHMWPE) particles on the osteogenic activity of primary murine bone marrow osteoprogenitors and MC3T3-E1 preosteoblastic cells in vitro. Submicron-sized UHMWPE particles generated from wear simulator tests were isolated from serum-containing solution by density gradient centrifugation. The particles were coated onto the surface of culture wells at concentrations of 0.038, 0.075, 0.150, 0.300, and 0.600% v/v in a layer of type I collagen matrix. Primary murine bone marrow cells and MC3T3-E1 preosteoblasts were seeded onto the particle-collagen matrix and induced to differentiate in osteogenic medium for 20 days. Exposure of both cell populations to UHMWPE particles resulted in a dose-dependent decrease in mineralization, proliferation, alkaline phosphatase activity, and osteocalcin production when compared with control cells cultured on collagen matrix without particles. Complete suppression of osteogenesis was observed at particle concentrations > or =0.150% v/v. This study demonstrated that UHMWPE particles inhibit the osteogenic activity of osteoprogenitor cells, which may result in reduced periprosthetic bone regeneration and repair.     

DKK1基因沉默的人多发性骨髓瘤细胞培养上清对小鼠前成骨细胞成骨分化的影响

 目的：初步探讨一种与胚胎发生及肿瘤发展密切相关的糖蛋白Dickkopf-1（DKK1）在骨髓瘤骨病（myeloma bone disease，MBD）发病中的作用。方法：前期实验构建的质粒pLenti6/V5-GW/DKK-1-miR，与慢病毒包装质粒一起包装成慢病毒，感染人骨髓瘤U266细胞，建立DKK1沉默的U266细胞。小鼠前成骨细胞MC3T3-E1体外成骨诱导实验分组：空白对照组、诱导分化组、30% U266上清干预组、30% DKK1 RNAi U266上清干预组和30%无关序列RNAi U266上清干预组；诱导培养12 d后，通过茜素红染色检测钙结节数目；real-time PCR检测骨钙素（osteocalcin，OC）mRNA的表达变化。结果：成骨诱导体系中，与空白组比较，诱导组OC mRNA有明显升高，差异有统计学意义（P<0.01）。与诱导组比较，30% U266上清干预组OC mRNA表达明显减少（P<0.01）。与30% U266上清干预组比较，30% DKK1 RNAi U266上清干预组OC mRNA表达明显增多（P<0.05），30%无关序列RNAi U266上清干预组OC mRNA差异无统计学意义。与诱导组比较，30% U266上清干预组钙结节计数明显减少（P<0.01）。与30% U266上清干预组比较，30% DKK1 RNAi U266上清干预组钙结节计数明显增多（P<0.05）,30%无关序列RNAi U266上清干预组钙结节计数差异无统计学意义(P>0.05)。结论：U266细胞中DKK1基因沉默后，其培养上清抑制小鼠前成骨细胞MC3T3-E1成骨分化的能力减弱。     

人牙周膜干细胞来源外泌体干预成骨细胞MC3T3-E1的增殖和分化

背景:人牙周膜干细胞具有较强的成骨分化能力,人牙周膜干细胞来源外泌体作为牙周膜干细胞分泌的主要成分,对成骨细胞MC3T3-E1增殖和成骨分化的影响尚不明确。目的:探讨人牙周膜干细胞来源外泌体对MC3T3-E1细胞增殖和分化的影响。方法:采用酶消化法分离及培养人牙周膜干细胞,超速离心法提取人牙周膜干细胞来源外泌体,通过透射电镜、粒径分析及Western blot方法对人牙周膜干细胞来源外泌体进行鉴定;CCK8法检测不同质量浓度人牙周膜干细胞来源外泌体对MC3T3-E1细胞增殖的影响,茜素红染色观察100 mg/L人牙周膜干细胞来源外泌体对MC3T3-E1细胞成骨矿化的影响,Western blot检测100 mg/L人牙周膜干细胞来源外泌体干预前后MC3T3-E1细胞内MEK和ERK的磷酸化水平。结果与结论:①透射电镜观察可见外泌体为脂质双分子层形成的囊泡结构,粒径检测显示外泌体直径分布在50-120 nm,集中在79.86 nm,Western blot检测结果显示提取的外泌体中含有CD81,CD63,TSG101的表达;②与对照组相比,人牙周膜干细胞来源外泌体对MC3T3-E1细胞的增殖具有促进作用,且作用呈剂量依赖性;③与对照组相比,人牙周膜干细胞来源外泌体组MC3T3-E1细胞能够形成更多的钙结节;与对照组相比,人牙周膜干细胞来源外泌体组MC3T3-E1细胞内p-MEK及p-ERK蛋白表达量升高;④结果表明,人牙周膜干细胞来源外泌体可以显著促进MC3T3-E1增殖和成骨分化,推测可能与其激活MEK/ERK信号通路有关。     

Novel nanostructured biodegradable polymer matrices fabricated by phase separation techniques for tissue regeneration

Biomimetic nanostructures have a wide range of applications. In particular, biodegradable polymer nanostructures that mimic the subtleties of extracellular matrix may provide favorable cell interactions. In this study, a co-solvent system was developed to configure a thermodynamically metastable biodegradable polymer solution, from which novel nanostructured matrices subsequently formed via wet phase separation (quaternary) or a combination with thermally induced phase separation. Three-dimensional (3D) nanostructured porous matrices were further fabricated by combination with particle-leaching (100–300 μm glucose). The new co-solvent system may generate matrices with reproducible nanostructures from a variety of biodegradable polymers such as poly(d,l-lactide) (PLA), poly(ε-caprolactone) (PCL) and PCL-based polyurethane. In vitro cell culture experiments were performed with mouse pre-osteoblasts (MC3T3-E1) and human bone marrow-derived mesenchymal stem cells (hBM-MSC) to evaluate the osteoinductive potential of PLA nanostructures. The results showed that nanofibrous (<100 nm) membranes promoted the bone-related marker gene expression and matrix mineralization of MC3T3-E1 at 14 days. Nanofibrous 3D matrices seeded with hBM-MSC without osteogenic induction supplements demonstrated a 2.5-fold increase in bone matrix deposition vs. the conventional microporous matrices after 14 and 21 days. Antimicrobial nanofibers were further obtained by plasma-assisted coating of chitosan on PLA nanofibers. This study reveals a platform for fabricating novel biodegradable nanofibrous architecture, with potential in tissue regeneration.     

骨形态发生蛋白2诱导成骨分化与长链非编码RNA AK007000的影响

背景：长链非编码RNA调控一系列生理过程,被认为在发育、分化和代谢的基因调控中发挥重要的作用。MC3T3-E1、C2C12和C3H10T1/2细胞可向骨细胞、肌细胞等多个方向分化,用于肌肉骨骼等运动系统相关疾病的研究。 目的：观察长链非编码RNA在骨形态发生蛋白2诱导成骨分化中的作用。 方法：对MC3T3-E1、C2C12和C3H10T1/2细胞在骨形态发生蛋白2诱导下,成骨分化过程中的长链非编码RNA表达变化进行芯片分析,找到在3株细胞中同时变化的长链非编码RNA,siRNA干扰方法观察长链非编码RNA对骨形态发生蛋白2诱导成骨分化的影响,采用Real-Time PCR与碱性磷酸酶染色检测成骨相关指标。 结果与结论：骨形态发生蛋白2诱导MC3T3-E1、C2C12和C3H10T1/2成骨分化过程中,相应成骨指标增高,成肌指标肌细胞生成素降低。筛选出骨形态发生蛋白2诱导成骨分化过程中出发挥作用的长链非编码RNA AK007000。AK007000被干扰后成骨分化指标碱性磷酸酶、骨钙素、RUNX2、SP7表达下降,肌细胞生成素表达上升。因此,AK007000可能具有促进成骨抑制成肌作用。 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：     

The effect of silica nanoparticle-modified surfaces on cell morphology, cytoskeletal organization and function

Chemical and morphological characteristics of a biomaterial surface are thought to play an important role in determining cellular differentiation and apoptosis. In this report, we investigate the effect of nanoparticle (NP) assemblies arranged on a flat substrate on cytoskeletal organization, proliferation and metabolic activity on two cell types, Bovine aortic endothelial cells (BAECs) and mouse calvarial preosteoblasts (MC3T3-E1). To vary roughness without altering chemistry, glass substrates were coated with monodispersed silica nanoparticles of 50, 100 and 300 nm in diameter. The impact of surface roughness at the nanoscale on cell morphology was studied by quantifying cell spreading, shape, cytoskeletal F-actin alignment, and recruitment of focal adhesion complexes (FAC) using image analysis. Metabolic activity was followed using a thiazolyl blue tetrazolium bromide assay. In the two cell types tested, surface roughness introduced by nanoparticles had cell type specific effects on cell morphology and metabolism. While BAEC on NP-modified substrates exhibited smaller cell areas and fewer focal adhesion complexes compared to BAEC grown on glass, MC3T3-E1 cells in contrast exhibited larger cell areas on NP-modified surfaces and an increased number of FACs, in comparison to unmodified glass. However, both cell types on 50 nm NP had the highest proliferation rates (comparable to glass control) whereas cells grown on 300 nm NP exhibited inhibited proliferation. Interestingly, for both cell types surface roughness promoted the formation of long, thick F-actin fibers, which aligned with the long axis of each cell. These findings are consistent with our earlier result that osteogenic differentiation of human mesenchymal progenitor cells is enhanced on NP-modified surfaces. Our finding that nanoroughness, as imparted by nanoparticle assemblies, effects cellular processes in a cell specific manner, can have far reaching consequences on the development of "smart" biomaterials especially for directing stem cell differentiation.     

流体剪应力对成骨分化不同阶段细胞Piezo1基因表达的影响

目的系统研究流体剪应力(fluid shear stress,FSS)作用下成骨分化方向不同阶段4种细胞内Piezo1离子通道蛋白的基因表达情况。方法利用自行研发的锥板流动腔技术,对小鼠来源的间充质干细胞MSC、成骨样细胞MC3T3-E1、成骨末期细胞MLO-A5和骨细胞MLO-Y4施加不同强度FSS(0.1、1.1 Pa),并分别在0、0.5、1、3、6、12 h等不同时间点抽提细胞内总RNA,应用实时荧光定量PCR技术研究FSS刺激对Piezo1基因表达的影响情况。结果 MSC、MC3T3-E1、MLO-A5和MLO-Y4细胞内都表达Piezo1和Piezo2。FSS作用后,所有细胞的Piezo1表达明显升高,并且1.1 Pa FSS刺激下的表达水平显著高于0.1 Pa。MSC、MC3T3-E1和MLO-A5这3种细胞在FSS刺激1 h后,Piezo1表达水平达到最高。Piezo1表达依赖于细胞种类,其中MC3T3-E1细胞的表达量高于其他3种细胞。结论 Piezo1的表达与成骨分化过程、FSS水平和加载时间相关,研究结果对于揭示骨组织细胞内力学信号的传递机制以及发展相应临床治疗措施具有重要意义。     

Polyurethane/nano-hydroxyapatite composite films as osteogenic platforms

A wide variety of biomaterials are utilized in tissue engineering to promote cell proliferations in vitro or tissue growth in vivo. The combination of cells, extracellular matrices, and biocompatible materials may make it possible to grow functional living tissues ranging from bone to nerve cells. In bone regeneration, polymeric scaffolds can be enhanced by the addition of bioactive materials. To this end, this study designed several ratios of polyurethane (PU) and nano-hydroxyapatite (nHA) composites (PU-nHA ratios: 100/0, 90/10, 80/20, 70/30, 60/40 w/w). The physical and mechanical properties of these composites and their relative cellular compatibility in vitro were determined. The chemical composition and crystallinity of the composites were confirmed using X-ray diffraction, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Atomic force microscopy, nano-indentation, and contact angle measurements were used to evaluate surface properties. The results showed a significant increase in surface roughness and a decrease in contact angle when the nHA concentration increased above 20%, resulting in a significant increase in hydrophilicity. These surface property changes influenced cellular behavior when MC 3T3-E1 cells were seeded on the composites. All composites were cytocompatible. There was a linear increase in cell proliferation on the 80/20 and 70/30 composites only, whereas subjective evaluation demonstrated noticeable clusters or nodules of cells (considered hallmarks of osteogenic differentiation) in the absence of any osteogenic inducers only on the 90/10 and 80/20 composites. Cellular data suggests that the 80/20 composite was an optimal environment for cell adhesion, proliferation, and, potentially, osteogenic differentiation in vitro.     

Co-culture with periodontal ligament stem cells enhanced osteoblastic differentiation of MC3T3-E1 cells and osteoclastic differentiation of RAW264.7 cells

Objectives: Periodontal ligament stem cells (PDLSCs) are characterized by having multipotential differentiation and immunoregulatory properties, which are the main mechanisms of PDLSCs-mediated periodontal regeneration. Periodontal or bone regeneration requires coordination of osteoblast and osteoclast, however, very little is known about the interactions between PDLSCs and osteoblast-like cells or osteoclast precursors. In this study, the indirect co-culture approach was introduced to preliminarily elucidate the effects of PDLSCs on differentiation of osteoblast-like cells and osteoclast precursors in vitro. Materials and methods: Human PDLSCs were obtained from premolars extracted and their stemness was identified in terms of their colony-forming ability, proliferative capacity, cell surface epitopes and multi-lineage differentiation potentials. A noncontact co-culture system of PDLSCs and preosteoblastic cell line MC3T3-E1 or osteoclast precursor cell line RAW264.7 was established, and osteoblastic differentiation of MC3T3-E1 and osteoclastic differentiation of RAW264.7 were evaluated. Results: PDLSCs exhibited features of mesenchymal stem cells. Further investigation through indirect co-culture system showed that PDLSCs enhanced ALP activity, expressions of ALP, Runx2, BSP, OPN mRNA and BSP, OPN proteins and mineralization matrix deposition in MC3T3-E1. Meanwhile, they improved maturation of osteoclasts and expressions of TRAP, CSTK, TRAF6 mRNA and TRAP, TRAF6 proteins in RAW264.7. Conclusions: PDLSCs stimulates osteoblastic differentiation of osteoblast precursors and osteoclastic differentiation of osteoclast precursors, at least partially, in a paracrine fasion.     

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.     

Asxl1在炎性微环境中对成骨细胞增殖分化的作用

背景:研究表明Asxl1的缺失可导致骨质发育不全、骨质缺损类疾病的发生,但目前在根尖周炎环境下该因子与骨破坏之间的关系暂无相关报道。目的:探讨炎性微环境下Asxl1对成骨细胞增殖分化的影响。方法:实验选用脂多糖刺激MC3T3-E1细胞建立体外炎性微环境,通过CCK-8实验筛取脂多糖最佳质量浓度和最佳作用时间,然后用20 mg/L脂多糖刺激MC3T3-E1细胞24 h,免疫荧光检测Asxl1的蛋白表达水平,Real Time-PCR检测Asxl1 mRNA的表达水平。为进一步验证Asxl1基因在炎性微环境中影响成骨细胞的增殖与分化,脂多糖刺激形成炎性微环境后转染Asxl1-SiRNA 24 h,采用CCK-8检测细胞增殖活性,RealTime-PCR检测Asxl1及成骨相关基因ALP和RUNX2 mRNA的表达水平。结果与结论:①脂多糖刺激MC3T3-E1细胞后,Asxl1蛋白和mRNA表达水平呈降低趋势;②脂多糖刺激MC3T3-E1细胞后,转染Asxl1-SiRNA 24 h,细胞增殖活性下降趋势明显,Asxl1基因及成骨相关基因ALP和RUNX2 mRNA的表达水平明显降低;③结果提示,Asxl1可能通过参与炎性反应过程,影响成骨细胞的增殖与分化,进而参与骨破坏进程。     

Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression

Human mesenchymal stem cells (hMSCs) differentiate down an osteogenic pathway with appropriate mechanical and/or chemical stimuli. This study describes the successful culture of hMSCs in 3D collagen matrices under mechanical strain. Bone marrow-derived hMSCs were seeded in linear 3D type I collagen matrices and subjected to 0%, 10%, or 12% uniaxial cyclic tensile strain at 1 Hz for 4 h/day for 7 or 14 days. Cell viability studies indicated that hMSCs remained viable throughout the culture period irrespective of the applied strain level. Real-time RT-PCR studies indicated a significant increase in BMP-2 mRNA expression levels in hMSCs strained at 10% compared to the same day unstrained controls after both 7 and 14 days. An increase in BMP-2 was also observed in hMSCs subjected to 12% strain, but the increase was significant only in the 14-day sample. This is the first report of the culture of bone marrow-derived hMSCs in 3D collagen matrices under cyclic strain, and the first demonstration that strain alone can induce osteogenic differentiation without the addition of osteogenic supplements. Induction of bone differentiation in 3D culture is a critical step in the creation of bioengineered bone constructs.     

Synthesis of biodegradable poly(ε-caprolactone)-organosiloxane hybrid with carboxylate groups

Biodegradable poly(ε-caprolactone)-organosiloxane hybrid with carboxylate groups was newly synthesized by a sol-gel method with the capacity to conjugate cell adhesion ligands for the potential applications as a bone tissue engineering scaffold material. An intermediate hybrid contained positively charged amine groups was synthesized by end-capping α,ω-hydroxyl poly(ε-caprolactone) with (3-isocyanatopropyl)triethoxysilane and a subsequent sol-gel reaction with (3-aminopropyl)triethoxysilane. This resulted in a new hybrid composed of biodegradable organic poly(ε-caprolactone) segments connected by inorganic siloxane linkages containing positively charged amine groups. The successive succinlylation of amine groups created amide bonds and converted the cationic sites of succinic anhydride into negatively charged carboxylate groups. New formations of amine and carboxylate groups could be directly and indirectly confirmed using Fourier transformed infrared spectrometry, zeta potentials, and atomic force microscopy. Biodegradability of hybrid with carboxylate groups was also examined by weight loss in phosphate buffered saline, and it was about 14 wt % after 8 weeks. Conjugating capacity of a cell adhesion ligand was examined by comparing the relative fluorescent intensities of covalently and noncovalently immobilized synthetic GGGGRGDASSK-FITC oligopeptides onto the carboxylated groups of the hybrid using confocal laser scanning microscopy; the relative fluorescent intensity of covalently conjugated RGD peptide was about three times higher than that of noncovalently coated RGD peptide. The hybrid covalently conjugated by GGGGRGDASSK oligopeptides improved proliferation and differentiation activities of preosteoblastic MC3T3-E1 cells. The results suggest that this hybrid possesses an encouraging potential to be used as a bone tissue engineering scaffold material due to its biodegradability and high capacity for conjugating cell adhesion ligands.     

MC3T3-E1 osteoprogenitor cells systemically migrate to a bone defect and enhance bone healing

Although iliac crest autologous bone graft remains the gold standard for treatment of bone defects, delayed- and nonunions, and arthrodeses, several alternative strategies have been attempted, including the use of mesenchymal stem cells. Whether cells from the osteoblast lineage demonstrate systemic recruitment to an acute bone defect or fracture, and whether these cells directly participate in bone healing is controversial. This study tests two hypotheses: (1) that exogenous murine MC3T3-E1 osteoprogenitor cells with a high propensity for osteoblast differentiation are able to systemically migrate to a bone defect and (2) that the migrated MC3T3-E1 cells enhance bone healing. Two groups of nude mice were used; a bone defect was drilled in the left femoral shaft in both groups. MC3T3-E1 were used as reporter cells and injected in the left ventricle of the heart, to avoid sequestration in the lungs. Injection of saline served as a control. We used bioluminescence and microCT to assay cell recruitment and bone mineral density (BMD). Immunohistochemical staining was used to confirm the migration of reporter cells. MC3T3-E1 cells were found to systemically migrate to the bone defect. Further, BMD at the defect was significantly increased when cells were injected. Systemic cell therapy using osteoprogenitor cells may be a potential strategy to enhance bone healing.     

骨形态发生蛋白2诱导C2C12和MC3T3-E1的成骨分化与自噬

背景：一系列研究表明自噬与分化有密切联系;骨形态发生蛋白2是诱导C2C12、MC3T3-E1成骨分化经典途径,是研究成骨分化过程的理想模型。 目的：观察自噬与骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化的关系。 方法：Real-Time PCR检测MC3T3-E1与C2C12在骨形态发生蛋白2(100 μg/L)诱导培养3 d后成骨与自噬相关指标变化。碱性磷酸酶染色检测不同浓度3-甲基腺嘌呤(0,1,5,10 mmol/L)对骨形态发生蛋白2(100 μg/L)诱导培养7 d MC3T3-E1与C2C12成骨指标碱性磷酸酶变化,Western Blot检测C2C12和MC3T3-E1在骨形态发生蛋白2(100 μg/L)诱导不同时间点(0,12,24,48,72,96 h)LC3-Ⅰ/Ⅱ蛋白表达水平。 结果与结论：在骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化过程中,诱导自噬相关mRNA与蛋白水平均有增高趋势,且自噬相关蛋白LC3水平增高与时间相关。同时,抑制自噬成骨分化过程中碱性磷酸酶表达水平降低。因此,自噬与骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化过程有密切关系。 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：     

Indirect coating of RGD peptides using a poly-L-lysine spacer enhances jaw periosteal cell adhesion, proliferation, and differentiation into osteogenic tissue

The aim of our study was to generate a biofunctionalized, three-dimensional (3D) biomaterial to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue. Therefore, open-cell polylactic acid (OPLA) scaffolds were coated covalently with different RGD peptides (a conserved recognition sequence of the most ECM proteins--arginine-glycine-asparagine) and different coating variants. The linear and cyclic RGD peptides were either applied directly or indirectly via a poly-L-lysine (PLL) spacer. JPCs were analyzed on coated constructs in 2D and 3D cultures and showed enhanced rates for indirectly coated scaffolds using the PLL spacer. By gene expression, we detected significantly increased levels of osteogenic marker genes, such as alkaline phosphatase, RUNX2, and AMELY in JPCs seeded onto PLL/linear RGD constructs compared to the otherwise-coated constructs. An analysis of the JPC mineralization capacity revealed the highest amounts of calcium-phosphate precipitates in cells growing within the PLL/linear scaffolds. Additionally, the JPC adhesion behavior on OPLA scaffolds seems to be mediated by ITGB3, ITGB1, and ITGAV, as shown by blocking assays. We concluded that coating of OPLA constructs with linear RGD peptides via PLL represents a suitable approach for functionalizing the polymer surface and enhancing adhesion, proliferation, and mineralization of JPCs.