Engineering human bone grafts with new macroporous calcium phosphate cement scaffolds

Bone engineering opens the possibility to grow large amounts of tissue products by combining patient‐specific cells with compliant biomaterials. Decellularized tissue matrices represent suitable biomaterials, but availability, long processing time, excessive cost, and concerns on pathogen transmission have led to the development of biomimetic synthetic alternatives. We recently fabricated calcium phosphate cement (CPC) scaffolds with variable macroporosity using a facile synthesis method with minimal manufacturing steps and demonstrated long‐term biocompatibility in vitro. However, there is no knowledge on the potential use of these scaffolds for bone engineering and whether the porosity of the scaffolds affects osteogenic differentiation and tissue formation in vitro. In this study, we explored the bone engineering potential of CPC scaffolds with two different macroporosities using human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC‐MP) or isolated from bone marrow (BMSC). Biomimetic decellularized bone scaffolds were used as reference material in all experiments. The results demonstrate that, irrespective of their macroporosity, the CPC scaffolds tested in this study support attachment, viability, and growth of iPSC‐MP and BMSC cells similarly to decellularized bone. Importantly, the tested materials sustained differentiation of the cells as evidenced by increased expression of osteogenic markers and formation of a mineralized tissue. In conclusion, the results of this study suggest that the CPC scaffolds fabricated using our method are suitable to engineer bone grafts from different cell sources and could lead to the development of safe and more affordable tissue grafts for reconstructive dentistry and orthopaedics and in vitro models for basic and applied research.     

Collagen scaffolds with in situ‐grown calcium phosphate for osteogenic differentiation of Wharton's jelly and menstrual blood stem cells

The aim of this research was to investigate the osteogenic differentiation potential of non‐invasively obtained human stem cells on collagen nanocomposite scaffolds with in situ‐grown calcium phosphate crystals. The foams had 70% porosity and pore sizes varying in the range 50–200 µm. The elastic modulus and compressive strength of the calcium phosphate containing collagen scaffolds were determined to be 234.5 kPa and 127.1 kPa, respectively, prior to in vitro studies. Mesenchymal stem cells (MSCs) obtained from Wharton's jelly and menstrual blood were seeded on the collagen scaffolds and proliferation and osteogenic differentiation capacities of these cells from two different sources were compared. The cells on the composite scaffold showed the highest alkaline phosphatase activity compared to the controls, cells on tissue culture polystyrene and cells on collagen scaffolds without in situ‐formed calcium phosphate. MSCs isolated from both Wharton's jelly and menstrual blood showed a significant level of osteogenic activity, but those from Wharton's jelly performed better. In this study it was shown that collagen nanocomposite scaffolds seeded with cells obtained non‐invasively from human tissues could represent a potential construct to be used in bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

糖皮质激素促间充质干细胞的成骨效应

背景：糖皮质激素是有效的免疫抑制和抗炎症药物而广泛应用于临床,然而长期服用后也会引起一些不良反应如骨质疏松症。糖皮质激素通过促进成骨细胞与骨细胞的凋亡,同时抑制间充质干细胞向成骨细胞分化,导致成骨细胞数量减少;然而在一定的条件下,糖皮质激素又能够促进间充质干细胞表达成骨细胞标志基因诱导其向成骨方向分化。目的：对以地塞米松为代表的糖皮质激素在间充质干细胞成骨分化中发挥的作用以及可能的分子机制进行综述。方法：由第一作者检索PubMed数据库1978至2012年有关地塞米松和间充质干细胞成骨分化的文献,英文关键词“Mesenchymal stem cell, dexamethasone, osteogenesis, osteoporosis, Runx2, BMP,Noggin, GILZ, glucocorticoid receptor”以不同的组合方式查找,排除重复性的研究,最终保留55篇进行归纳综述。结果与结论：地塞米松能够调节Runx2,Noggin以及亮氨酸拉链蛋白等基因调控间充质干细胞成骨分化。当糖皮质激素作用于间充质干细胞时,可能由糖皮质激素受体介导其生理作用,也可能在糖皮质激素与其受体结合之前就受到11β-羟甾类脱氢酶的调节。另外,生理剂量（10^-88mol／L）的地塞米松能促进问充质干细胞成骨分化,药理剂量（≥10^-7mol／L）有抑制作用。     

Bone differentiation of marrow-derived mesenchymal stem cells using beta-tricalcium phosphate-alginate-gelatin hybrid scaffolds

The aim of the present study was to establish a 3D culture system for bone differentiation of mesenchymal stem cells (MSCs), using a new hybrid sponge. To manufacture the scaffold, a composite of beta-tricalcium phosphate-alginate-gelatin was prepared and cast as pellets of 1 cm diameter. The sponge was then fabricated by drying in freeze-dryer for 12 h. The porosity, mean pore size, compressive modulus and strength of the composite sponge fabricated in this study were 89.7%, 325.3 microm, 1.82 and 0.196 MPa, respectively. To establish a 3D culture system, the rat bone marrow-derived MSCs were suspended in 500 microl diluted collagen gel, loaded into the porous sponge and provided with medium with or without osteogenic supplements for 3 weeks. The day after loading, the cells appeared in the scaffold's internal spaces, where later some of them from either culture survived by anchoring on the surfaces. At the end of cultivation period, individually adhered cells from both cultures were observed to be replaced by cell aggregates, in which mineralized matrix was detected by alizarin red staining. Furthermore, RT-PCR analysis indicated that the bone-specific gene osteocalcin was expressed in cultures in both the presence and absence of the osteogenic supplements. Taken together, it seems that the studied scaffolds are cell-compatible and, more importantly, possess some osteo-inductive properties.     

Effect of mechanical loading and substrate elasticity on the osteogenic and adipogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are highly sensitive to biomechanics of their extracellular environment. Generally, a higher elasticity of culture substrates can drive cells into the osteogenic lineage, whereas low substrate elasticity results in adipogenesis. Applied mechanical loading by cyclic strain is another major variable influencing cell fate. Yet, little is known about the simultaneous effect of both cues. Therefore, the present study investigated the relative importance of both cues on differentiation. MSCs were cultured in an osteogenic and also an adipogenic environment on soft polyacrylamide (PAAm; E = 23 ± 0.3 kPa), stiff PAAm (111 ± 2 kPa), and polydimethylsiloxane (PDMS; E = 1,5 ± 0.07 MPa) either unstrained or with 8% cyclic strain at 1 Hz. Without strain, the relative expression of the early osteogenic marker alkaline phosphatase (ALP) was significantly higher (78%) on PDMS than on both PAAm. With 8% cyclic strain, ALP expression increased for all groups in comparison with unstrained controls. The highest increase was observed for the soft PAAm by 36%. Moreover, relative oil red O (ORO) expression—indicating adipogenesis—was the highest for unstrained soft PAAm. On the other hand, the percentage of ORO positive cells significantly decreased by 57% and 69% for soft and stiff PAAm when strained. In conclusion, biomaterial elasticity and mechanical loading can act simultaneously on cell differentiation. Substrate elasticity is an important factor, regulating the differentiation, but cyclic strain can drive MSCs towards the osteogenesis even on the softest substrate. As such, the osteogenic effect of mechanical loading can overrule the adipogenic effect of soft substrates, thereby acting as an inhibitor.     

Differential bone‐forming capacity of osteogenic cells from either embryonic stem cells or bone marrow‐derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue‐engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune‐deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied. Copyright © 2010 John Wiley & Sons, Ltd.     

脂肪间充质干细胞复合骨诱导性磷酸钙陶瓷支架的异位成骨☆

背景：生物材料的骨诱导现象已经在多种动物实验中被证实。目的：考察磷酸钙陶瓷自身固有的诱导骨生成能力在其作为骨组织工程支架时的表现。方法：取健康家犬10只,在每只的背部肌肉内分别植入骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物、非骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物、骨诱导性磷酸钙陶瓷及非骨诱导性磷酸钙陶瓷,植入后8,12周,取出植入材料及其周围组织进行Micro-CT检测和组织形态学检测,评价成骨情况。结果与结论：组织学观察结果显示,骨诱导性磷酸钙陶瓷组及骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物组均有有异位骨生成,并且骨诱导性磷酸钙陶瓷与自体脂肪间充质干细胞复合物组的成骨量显著大于骨诱导性磷酸钙陶瓷组(P<0.05);其余两组均无异位成骨。Micro-CT检测结果与组织形态学检测结果一致。结果表明骨诱导性磷酸钙陶瓷作为骨组织工程支架材料有明显的成骨优势,而脂肪间充质干细胞作为种子细胞对异位成骨有明显的促进作用。     

Simvastatin induces osteogenic differentiation in human amniotic fluid mesenchymal stem cells (AFMSC)

Amniotic fluid is a complex mixture composed of water, salts and different cells types derived from embryo exfoliation. Some of these cells present similar characteristics to mesenchymal stem cells as adherent properties, typical surface antigens and differentiation capacity. These cells are called amniotic fluid‐derived mesenchymal stem cells (AFMSCs) and are easily obtained by amniocentesis, propagated in culture and differentiated in several cell types with specific inductions. In this study, we observe the ability of simvastatin, a 3‐HMG‐CoA reductase inhibitor, to induce AFSMCs osteogenic differentiation. When AFSMCs were incubated with medium containing simvastatin, it was observed morphological changes, calcium deposits formation confirmed by Alizarin Red stain. Differentiated cells also expressed typical osteogenic genes, as osteopontin and osteocalcin. In conclusion, simvastatin could be used as an optional osteogenic induction agent for amniotic fluid‐derived mesenchymal stem cells.     

人骨髓间充质干细胞向成骨细胞分化中26RFa的作用

背景：研究表明26RFa在骨形成、痛觉、内分泌、心血管以及能量代谢等方面都有重要的调节作用。目的：观察26RFa在成骨培养体系中对人骨髓间充质干细胞增殖分化的影响。 方法：通过MTT实验,以此确定26RFa对人骨髓间充质干细胞是否有促增殖作用及其起作用的最大活性浓度;将人骨髓间充质干细胞接种于6孔培养板上,实验分为2组：实验组含有10-11mol/L的26RFa,对照组不含26RFa。取成骨诱导8,12,16 d细胞用碱性磷酸酶试剂盒测定细胞内碱性磷酸酶活性;成骨诱导21,28 d后行茜素红染色和Von Kossa染色,计算每张玻片钙化结节数,Western blot检测cbfa1的表达。 结果与结论：成骨诱导8,12,16 d后细胞内碱性磷酸酶活性实验组高于对照组（P〈0.05,P〈0.01,P〈0.05）;21,28 d后茜素红染色和Von Kossa矿化结节数实验组均高于对照组;实验组细胞cbfa1表达明显高于对照组（P〈0.05）。说明在适宜的培养条件下,26RFa可促进人骨髓间充质干细胞向成骨细胞分化。     

全骨髓贴壁法培养兔骨髓间充质干细胞体外定向成骨诱导分化及鉴定

背景：流式细胞仪分离法和免疫磁珠分离法对细胞活性影响较大,密度梯度离心法虽然能够获得纯度高的单核细胞,但由于多次离心可造成细胞的大量流失且对细胞活性有一定的影响使其应用值得商榷。目的：采用全骨髓贴壁法分离兔骨髓间充质干细胞进行成骨诱导分化及鉴定。方法：采用全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,倒置显微镜下观察细胞形态学特征。在成骨诱导剂作用下,通过碱性磷酸酶染色试剂盒行碱性磷酸酶染色,I型胶原免疫细胞化学染色,VonKossa法及茜素红进行矿化结节染色以及电镜下检测兔骨髓间充质干细胞成骨诱导后的形态结构。结果与结论：经诱导后细胞出现与成骨细胞相似的形态学特征,碱性磷酸酶染色阳性,I型胶原免疫细胞化学染色,Von-Kossa法及茜素红矿化结节染色阳性。表明经成骨诱导剂诱导后全骨髓贴壁法体外分离纯化培养的兔骨髓间充质干细胞能向成骨细胞方向分化增殖。     

α- 玉米赤霉醇影响小鼠骨髓间充质干细胞的成骨分化简

背景：骨髓间充质干细胞具有向成骨细胞分化的能力,植物雌激素α-玉米赤霉醇对小鼠骨髓间充质干细胞的成骨分化可能有促进作用。 目的：探讨α-玉米赤霉醇对小鼠骨髓间充质干细胞向成骨细胞分化的影响。 方法：采用全骨髓培养差速贴壁法体外分离培养小鼠骨髓间充质干细胞,取第3代细胞分为6组：非诱导组加含体积分数为10%胎牛血清、1%双抗的 IMDM 普通培养基;空白诱导组仅加入等量成骨条件培养基（含0.1μmol/L 地塞米松、10 mmol/Lβ-甘油磷酸钠、50μmol/L 维生素C）;阳性对照诱导组加入等量成骨条件培养基及10-8 mol/L雌二醇;高、中、低α-玉米赤霉醇组分别加入成骨条件培养基及10-5,10-6,10-7 mol/L梯度浓度的α-玉米赤霉醇。倒置显微镜下观察细胞形态变化,MTT 实验测定细胞增殖状态绘制细胞生长曲线,酶联免疫吸附法测定碱性磷酸酶、骨钙素的表达。 结果与结论：非诱导组细胞呈长梭形,生长密集时有接触抑制,各诱导组的细胞增殖受促进,诱导后细胞呈三角形、多角形、不规则形状,细胞可重叠生长;非诱导组低表达碱性磷酸酶、骨钙素,低浓度（10-7 mol/L）α-玉米赤霉醇组和阳性对照诱导组高表达碱性磷酸酶、骨钙素,与空白诱导组相比,差异有非常显著性意义（P＜0.01）。结果说明α-玉米赤霉醇可以促进骨髓间充质干细胞的增殖,低浓度α-玉米赤霉醇可显著促进体外培养的小鼠骨髓间充质干细胞成骨分化。而上调碱性磷酸酶、骨钙素的表达量可能是α-玉米赤霉醇诱导促进骨髓间充质干细胞骨向分化的作用机制之一。     

Timing,rather than the concentration of cyclic AMP,correlates to osteogenic differentiation of human mesenchymal stem cells

Previously, we demonstrated that protein kinase A (PKA) activation using dibutyryl‐cAMP in human mesenchymal stem cells (hMSCs) induces in vitro osteogenesis and bone formation in vivo. To further investigate the physiological role of PKA in hMSC osteogenesis, we tested a selection of G‐protein‐coupled receptor ligands which signal via intracellular cAMP production and PKA activation. Treatment of hMSCs with parathyroid hormone, parathyroid hormone‐related peptide, melatonin, epinephrine, calcitonin or calcitonin gene‐related peptide did not result in accumulation of cAMP or induction of alkaline phosphatase (ALP) expression. The only ligand that did induce cAMP, prostaglandin E2, even inhibited ALP expression and mineralization, suggesting that physiological levels of cAMP may inhibit osteogenesis. Furthermore, intermittent exposure of hMSCs to dibutyryl‐cAMP inhibited ALP expression, whereas we did not observe an inhibitive effect at low dibutyryl‐cAMP concentrations. Taken together, our results demonstrate that cAMP can either stimulate or inhibit osteogenesis in hMSCs, depending on the duration, rather than the strength, of the signal provided. Copyright © 2009 John Wiley & Sons, Ltd.     

Fibronectin stimulates the osteogenic differentiation of murine embryonic stem cells

Conditioned medium from human hepatocarcinoma cells (HepG2‐CM) has been shown to stimulate the osteogenic/chondrogenic differentiation of murine embryonic stem cells (mESCs). HepG2‐CM is considered to contain visceral endoderm (VE)‐like signals and attempts have recently been made to characterize it, using proteomic profiling, with fibronectin being identified as one promising candidate. Herein, we investigated whether fibronectin is able to mimic the activities of HepG2‐CM during the osteogenic differentiation of mESCs. Specifically, the addition of RGD peptides and heparin in HepG2‐CM significantly reduced the growth‐ and adhesion‐promoting effects of HepG2‐CM, in addition to suppressing its osteogenic‐inductive activity. Furthermore, direct addition of fibronectin to basal medium was able to reproduce, at least partially, the function of HepG2‐CM. In particular, fibronectin induced the early onset of osteogenic differentiation in mESCs, as confirmed by gene expression of osteogenic markers, and resulted in the three‐fold higher calcium deposition at day 11 of osteogenic culture compared to the control group. These data clearly suggest that fibronectin contributes to the biological activities of HepG2‐CM and plays a stimulatory role during the process of osteogenesis in mESCs. Copyright © 2015 John Wiley & Sons, Ltd.     

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright © 2008 John Wiley & Sons, Ltd.     

淫羊藿对骨髓间充质干细胞成骨分化的影响简

背景：中药淫羊藿一直以来被用于骨质疏松的治疗和骨缺损的修复。目的：探讨淫羊藿对骨髓间充质干细胞成骨性分化的影响。 方法：应用数据库文献检索的方法获取淫羊藿对骨髓间充质干细胞成骨性分化影响研究的文献,对符合研究标准的文献进行深入的分析。通过检测淫羊藿诱导骨髓间充质干细胞成骨性分化过程中碱性磷酸酶、骨钙素、骨桥蛋白、转化生长因子β、骨形态发生蛋白、骨钙素、骨涎蛋白等,了解淫羊藿对骨髓间充质干细胞的增殖和成骨分化的机制。结果与结论：淫羊藿对骨髓间充质干细胞的影响呈剂量依赖性,在诱导培养的早期淫羊藿苷可以提高细胞的磷酸酶活性,在诱导培养的晚期增加细胞钙化结节数,促进骨钙素分泌量,显著提高转化生长因子β1、骨形成蛋白2、胰岛样生长因子1、骨桥蛋白和骨涎蛋白等的表达水平。淫羊藿对骨髓间充质干细胞的增殖和成骨性分化有促进作用,可作为一种良好的骨诱导活性因子。     

In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow‐derived cells were CD11b^–CD34^–CD44⁺CD45^–CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM + VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE‐cadherin, PECAM‐1, VEGF‐R1 and VEGF‐R2 after 10 days of stimulation. Cells cultured in EGM + VEGF medium demonstrated higher amounts of DiI‐AcLDL‐positive cells and enhanced the presence of vWF (90%), VE‐Cadherin‐ (60%) and PECAM‐1 (48%)‐positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2‐fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM + VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re‐endothelialization following interventional procedures and tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

不同因子影响骨髓间充质干细胞的多向分化

背景：在组织工程领域,关于骨髓间充质干细胞定向诱导分化的研究越来越多,但是细胞培养基中不同成分会对骨髓间充质干细胞的体外增殖分化产生影响。目的：针对培养基中不同因子对骨髓间充质干细胞定向诱导分化的作用加以综述。方法：第一作者应用计算机检索1998年1月至2012年4月Pubmed数据库及万方数据库。检索英文关键词为“bone marrow mesenchymal stromal cells, cell culture medium, differentiation”,中文关键词为“骨髓间充质干细胞,细胞培养,定向诱导分化”,纳入有关不同因子对骨髓间充质干细胞向成骨细胞、软骨细胞和脂肪细胞定向诱导分化作用的文献,排除重复研究。结果与结论：计算机初检共得到184篇文献,根据纳入排除标准,对其中30篇文献进行综述。大体说来,骨髓间充质干细胞向成骨分化的主要因子有地塞米松、转化生长因子、维生素C、维生素D3、β-甘油磷酸钠及乙烯雌酚等;向软骨分化的主要因子有地塞米松、转化生长因子、维生素C、胰岛素样生长因子及成纤维细胞生长因子等;向脂肪细胞转化的主要因子有地塞米松、3-异丁基-1-甲基黄嘌呤、胰岛素和消炎痛等,但是其中一些因子的作用机制及不良反应还不明确,需要进一步的研究与验证。同时,骨髓间充质干细胞在骨髓中的含量较低,不同的分离方法会导致不同的分离率,因此如何选取一种分离率高的分离方法仍有待研究。