Minimal perfusion flow for osteogenic growth of mesenchymal stem cells on lattice scaffolds

A modeling approach to identify sets of culture conditions to promote homogeneous growth of cells in perfusion bioreactors equipped with regular shape scaffolds is proposed. We identify cases in which dynamic culturing is necessary using a zero‐dimensional mass transport and reaction model. Then, based on the three‐dimensional (3‐D) rendering of the flow field inside the bioreactor, we identify regions where cellular growth may become critical; finally, using a 1‐D mass transport and reaction model, we calculate the minimal perfusion flow necessary to maintain the cellular growth rate above a target threshold. The developed approach is used to analyze culturing conditions inside an indirect perfusion bioreactor equipped with a lattice scaffold. Regions where the perfusion flow is inadequate to foster cellular growth at the desired rate are identified. The perfusion flow required to maintain the target growth rate inside the bioreactor is calculated. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3131–3144, 2013     

Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells

Graphene nanogrids (fabricated by graphene nanoribbons obtained through oxidative unzipping of multi-walled carbon nanotubes) were used as two-dimensional selective templates for accelerated differentiation of human mesenchymal stem cells (hMSCs), isolated from umbilical cord blood, into osteogenic lineage. The biocompatible and hydrophilic graphene nanogrids showed high actin cytoskeleton proliferations coinciding with patterns of the nanogrids. The amounts of proliferations were found slightly better than proliferation on hydrophilic graphene oxide (GO) sheets, and significantly higher than non-uniform proliferations on hydrophobic reduced graphene oxide (rGO) sheets and polydimethylsiloxane substrate. In the presence of chemical inducers, the reduced graphene oxide nanoribbon (rGONR) grid showed a highly accelerated osteogenic differentiation of the hMSCs (a patterned differentiation) in short time of 7 days in which the amount of the osteogenesis was ∼2.2 folds greater than the differentiation (a uniform differentiation) on the rGO sheets. We found that although in the absence of any chemical inducers the graphene nanogrids showed slight patterned osteogenic differentiations, the graphene sheets could not present any differentiation. Therefore, the highly accelerated differentiation on the rGONR grid was assigned to both its excellent capability in adsorption of the chemical inducers and physical stresses induced by the surface topographic features of the nanogrids.     

大鼠骨髓间充质干细胞向成骨与成脂分化过程中相关基因的表达变化

目的探讨大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)向成骨与成脂分化过程中相关基因表达的变化。方法采用全贴壁法分离培养大鼠BMSCs,并观察其形态学特征的变化,MTT法检测其生长状况,并绘制生长曲线。分别采用成骨和成脂诱导剂对第4代BMSCs进行诱导分化,应用碱性磷酸酶试剂盒、茜素红和油红O染色液检测其ALP活性、成骨和成脂分化能力;RT-QPCR检测诱导0、7、14和21 d的成骨分化相关基因Runt相关转录因子2(Runx2)、骨钙素(osteocalcin,OCN)、碱性磷酸酶(ALP)及成脂分化相关基因过氧化物酶体增殖物激活受体γ(peroxidase proliferator activated receptor gamma,PPARγ)和脂肪酸结合蛋白(FABP4)的表达变化。结果全骨髓贴壁法能成功分离培养BMSCs,传代细胞生长增殖迅速,以长梭形细胞生长为主,细胞生长曲线呈S形。第4代BMSCs分别经成骨和成脂诱导剂诱导后,ALP、茜素红和油红O染色均呈阳性;诱导7、14和21 d后,Runx2、OCN、ALP、PPARγ和FABP4基因mRNA的表达量均显著高于0 d(P0.05);成骨分化过程中,Runx2和ALP在第7天时表达量最高,之后呈下降趋势,OCN的表达量呈稳定上升趋势;成脂分化过程中,PPARγ在第7天时表达量最高,FABP4始终高表达。结论 BMSCs具有易于体外分离培养、扩增和经诱导后具有多向分化潜能等特点,成骨和成脂分化相关基因的表达量随诱导时间延长而变化,呈明显的时序性表达差异,提示分别在成骨与成脂分化过程中起重要调控作用,为BMSCs在骨、细胞和基因等工程中的机制研究提供了实验依据。     

Calcium phosphates Chitosan-Xanthan composite scaffolds associated with mesenchymal stem cells for regenerative dentistry application

The aim of this study was to synthesize and characterize polymeric porous scaffolds associated with different calcium phosphates (CaP) and Mesenchymal Stem Cells (MSC) for regenerative dentistry application. Chitosan-Xanthan Scaffolds (CX) were associated with 5% of the two CaP types, Hydroxyapatite (HA) and Brushite (BS). For advanced cell therapies, the scaffolds were associated with MSC. The scaffold structures were characterized by X Ray Diffraction (DRX), Fourier Transformed Infrared (FTIR), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy analysis (SEM) and the compressive strength. The in vitro Cytotoxicity was performed and the in vivo Biocompatibility by histomorphometry and inflammatory cells number count. XRD showed the amorphous phase of CX and main peaks of the CaP phases in the HA and BS scaffolds. FTIR showed the amide I and II bands, characteristic of Chitosan, and carboxyl group, characteristic of Xanthan. PO4 bands were found in CaP scaffolds. SEM showed pores and CaP fillers incorporated and adhered to the polymer in the CX-HA, CX-BS, CX-HA + MSC and CX-BS + MSC. Compressive strength and Modulus of Elasticity analysis exhibited higher values for CX-BS scaffolds, followed by CX-HA and CX. All scaffolds showed acceptable cells viability after 24 h and 48 h; however, the CX scaffolds showed higher cell viability in 48 h. CX-BS produced significantly higher inflammatory cells number after 7 and 30 days of implantation. After 60 days of implantation, CX + MSC and CX-HA + MSC showed the lowest inflammatory cells number. The CaP improved the mechanical properties of scaffolds but decreased the cell viability. MSCs improved the inflammatory response after 60 days.     

Electrospun nanofibrous alginate sulfate scaffolds promote mesenchymal stem cells differentiation to chondrocytes

Cartilage tissue engineering is one of the interesting approaches used for repairing cartilage injuries. This study reports the fabrication of polyvinyl alcohol/alginate sulfate (PVA/ALG-S) nanofibrous mats as a functional support for chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). The PVA/ALG-S nanofibers were obtained through electrospinning of PVA solutions containing 10, 20, and 30 wt% of ALG-S. The appearance of a band at 833 cm⁻¹ assigned to the symmetrical C O S vibration associated to a C O SO₃ group confirmed the presence of ALG-S in nanofibrous mat. The SEM images illustrated the bead-free and smooth morphology of PVA/ALG-S nanofibers with a mean diameter of 185 ± 0.06 nm. The MTT assay of the hBM-MSCs seeded on scaffolds indicated the appropriate cytocompatibility of nanofibrous PVA/ALG-S scaffolds. Furthermore, the appropriate attachment and spreading of the hBM-MSCs based on SEM images, and their differentiation to the chondrocyte-like cells accompanied by a decrease in cell growth on MTT analysis and more color absorption in alician blue staining indicated the effective role of alginate sulfate on cell differentiation. Finally, the expression of Type II collagen by RT-PCR and immunocytochemistry analyses revealed the chondrogenic differentiation of hBM-MSCs on alginate sulfate nanofibers.     

Evaluation of the chondrogenic differentiation of mesenchymal stem cells on hybrid biomimetic scaffolds

Hybrid materials are widely and promisingly used as scaffolds in cartilage tissue remodeling. In this study, hybrid scaffolds consist of polycaprolactone (PCL), poly(vinyl alcohol) (PVA) with/without gelatin (GEL) to mimic natural cartilage extracellular matrix (ECM) were investigated. Scaffolds were prepared by freeze drying and characterized by scanning electron microscopy and compressive mechanical testing. Biological assays of mesenchymal stem cell (MSC) cultures, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, and dimethyl methylene blue were performed, and real‐time polymerization chain reaction analysis of the cartilage‐specific ECM gene marker expression was done. The results show an open interconnected porous structure with a compression modulus of 1.27 ± 0.04 MPa. The surface of the scaffolds showed an excellent efficiency in the adhesion and proliferation of MSCs. A significant increase in the proteoglycan content from 3.70 ± 0.96 to 5.4 ± 1.13 μg/mL was observed after 14 days in the PCL–PVA–GEL scaffolds. The expression amount of the sex‐determining region Y–Box 9 (SOX9) and collagen II (COL2) mRNA levels of the MSCs showed significant increases in SOX9 and COL2, respectively in comparison with PCL–PVA scaffold. The study revealed that the aforementioned scaffold as a blend of natural and synthetic polymers may be a promising substrate in tissue engineering for cartilage repair with MSC transplantation. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40635.     

cAMP-PKA-CREB信号通路在骨形态发生蛋白9诱导小鼠间充质干细胞成骨分化中的作用

目的探讨cAMP-PKA-CREB信号通路在骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导小鼠间充质干细胞(mesenchymal stem cells,MSCs)C3H10T1/2成骨分化过程中的作用及其机制。方法将C3H10T1/2细胞分别加入不同浓度的cAMP-PKA-CREB信号通路抑制剂H89(1、2.5、5和10μmol/L),检测其对碱性磷酸酶(alkaline phosphatase,ALP)活性的影响;通过ALP定量和钙盐沉积试验分别检测H89对BMP9诱导C3H10T1/2细胞早期和晚期成骨分化的影响;经Western blot法检测H89对C3H10T1/2细胞中磷酸化CREB、骨钙素(Osteocalcin,OCN)和成骨关键转录因子Runx2表达水平的影响;通过Wentern blot及荧光素酶活性的检测,观察H89对经典信号通路BMPs-smad1/5/8的影响。结果随着H89浓度的增加,对BMP9诱导的C3H10T1/2细胞ALP的抑制作用明显增强(P0.05),且呈剂量依赖性;ALP定量和钙盐沉积试验结果表明,H89可明显抑制BMP9诱导的C3H10T1/2细胞早期及晚期成骨分化;H89可显著抑制BMP9诱导的C3H10T1/2细胞中磷酸化CREB、OCN及Runx2蛋白的表达(P0.05),与AdBMP9组比较,H89对经典BMPs-smad1/5/8信号通路无明显影响(P0.05)。结论阻断cAMP-PKA-CREB信号通路可抑制BMP9诱导的MSCs C3H10T1/2的成骨分化,为BMP9的临床应用奠定了理论基础。     

Protein expression by bone mesenchymal stem cells cultivated in PLLA scaffolds with different pore geometry

Abstract

The effects of poly(L,L-lactide) (PLLA) scaffold with axial and isotropic structure were investigated on functional activity of rabbit bone mesenchymal stem cells (BMSCs). PLLA scaffolds were processed by freeze-dry technique at different temperatures of the scaffold frost – ?196?°C, ?25?°C and 0?°C. Scaffolds with different pore sizes were obtained by adding 5 or 10% of water phase. Scaffolds were modified by collagen type I solution. The pore sizes of polymer scaffolds were ranging from 5 to 150?µm. More protein secretion was observed in the surface-modified scaffolds than in the unmodified after 2 weeks of cultivation in vitro.     

Cellular behavior of human mesenchymal stem cells cultured on single-walled carbon nanotube film

The influences of carboxylic functionalized single-walled carbon nanotubes (SWCNTs) on cell adhesion, spreading and cell lineage commitment of human mesenchymal stem cells (hMSCs) were evaluated. hMSCs were cultured on a thin mesh like layer of SWCNTs with a vertical height of less than 100 nm. The influence of the SWCNT film was significant on the cell spreading and focal adhesion distribution. Cells spread better on a SWCNT film as compared to cover slip (control), resulting in larger cell area and have higher occurrence of filopodia (microspikes) at the cell boundaries. Cytoskeleton arrangement was observed to be less orientated in the cells cultured on a SWCNT film as compared to control. Neurogenic markers such as nestin, glial fibrillary acidic protein and microtubule associated protein 2 genes were transiently upregulated (a process where cellular components, in this case RNA, is increased in response to external variable) over the first week while genes indicative of osteogenesis remained at its nominal level. These results suggest that nano roughness alone is sufficient to modulate cellular behavior and early stage of stem cell lineage commitment without the aid of an induction medium.     

Hepatic differentiation of rat mesenchymal stem cells by a small molecule

Mesenchymal stem cells (MSCs) are capable of self-renewal and multilineage differentiation. A periodic acid-Schiff (PAS) stain-based assay was developed to screen for small-molecule inducers of hepatic differentiation of bone marrow MSCs. 2-(4-Bromophenyl)-N-(4-fluorophenyl)-3-propyl-3H-imidazo[4,5-b]pyridin-5-amine (SJA710-6) was identified as a novel small molecule able to induce the differentiation of rat MSCs (rMSCs) toward hepatocyte-like cells in vitro, where rMSCs treated with SJA710-6 have typical morphological and functional characteristics of hepatic cells, including glycogen storage, urea secretion, uptake of low density lipoprotein (LDL) and expression of hepatocyte-specific genes and proteins. Expression of FoxH1 (FAST1/2) induces the differentiation of rMSCs towards hepatocyte-like cells, suggesting that this gene plays an important role in the hepatic fate specification of rMSCs.     

The effect of curvature on chondrocytes migration and bone mesenchymal stem cells differentiation

Numerous cells grow in columnar tissues and organs with different curvatures and curvature gradients. Therefore, it is necessary to study the effect of curvature on cell behavior to control and promote cell development. Herein, we prepared polydimethylsiloxane (PDMS) with different micro-nano patterns using ultraviolet soft lithography. Hydrophilic polydopamine (PDA) was modified on the PDMS surface to prepare PDMS/PDA to improve its biocompatibility. The PDMS/PDA was characterized by contact angle tester and scanning electron microscopy (SEM). The effect of curvature on bone cell migration and differentiation was studied through SEM, inverted phase contrast microscope and fluorescence microscopy. We found that different curvatures had different effects on the bone cell migration and differentiation. Chondrocytes migrated rapidly in grooves with a curvature range of 1/575–1/875 μm⁻¹. Bone mesenchymal stem cells (BMSCs) had high efficiency of differentiation into chondrocytes in the grooves with a curvature range of 1/775–1/1375 μm⁻¹. Furthermore, BMSCs showed high efficiency of differentiation into chondrocytes at the edges of micro-nano patterns with different perimeter curvatures, and the differentiation efficiency was the highest at 120° convex curvature. This work shows that curvature is a principle to be considered in bone tissue regeneration engineering and provides inspiration for future biomaterials design.     

Cobalt-polypyrrole-multiwalled carbon nanotube catalysts for hydrogen and alcohol fuel cells

A cobalt-polymer-MWCNT composite has been developed as an electrocatalyst for the oxygen reduction reaction (ORR) in PEMFC. The suitability of this electrocatalyst for the ORR in direct methanol fuel cells and direct ethanol fuel cells has been examined by taking Pt-Ru/MWCNT and Pt-Sn/MWCNT, respectively as an anode electrocatalysts. The study results in improved power densities for hydrogen, methanol and ethanol based fuel cells compared to the previously reported non Pt based electrocatalysts, highlighting the use of this cobalt-polymer-MWCNT composite as a potential candidate for ORR in fuel cells.     

Micro-/nano- sized hydroxyapatite directs differentiation of rat bone marrow derived mesenchymal stem cells towards an osteoblast lineage

Regenerative medicine consisting of cells and materials provides a new way for the repair and regeneration of tissues and organs. Nano-biomaterials are highlighted due to their advantageous features compared with conventional micro-materials. The aim of this study is to investigate the effects of micro-/nano- sized hydroxyapatite (μ/n-HA) on the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). μ/n-HA were prepared by a microwave synthesizer and precipitation method, respectively. Different sizes of μ/n-HA were characterized by IR, XRD, SEM, TEM and co-cultured with rBMSCs. It was shown that rBMSCs expressed higher levels of osteoblast-related markers by n-HA than μ-HA stimulation. The size of HA is an important factor for affecting the osteogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.     

The influence of strontium release rate from bioactive phosphate glasses on osteogenic differentiation of human mesenchymal stem cells

Strontium-doped bioactive phosphate glasses (P-glasses) of general composition 40(P₂O₅)–25(CaO)–5(Na₂O)–(30-x)MgO–x(SrO) (x = 0,1,5,10) were fabricated via melt-quenching technique and effects of strontium (Sr) content on P-glass properties were systematically studied, including glass structure and density, thermal properties, solubility and cytocompatibility with human mesenchymal stem cells (hMSCs). Sr-doping resulted in the weakening of glass network and a decrease in glass transition and melting temperatures. P-glasses maintained physiological pH on immersion in simulated body fluid and exhibited sustained ion release for up to ten weeks. All P-glasses tested were cytocompatible with hMSCs. Sr²⁺ release upregulated the genes associated with hMSC osteogenic differentiation; expression levels of early markers (RUNX2, COL 1) were proportional to the amount of Sr²⁺ in the P-glasses, while expression of late markers (AlP, OC) was the highest for the P-glass containing 5 mol% Sr. It was concluded that Sr-doped P-glasses promoted osteogenic differentiation of hMSCs and have considerable potential for bone tissue regeneration.     

去上皮羊膜及其浸提液体外诱导骨髓间充质干细胞的分化

目的观察去上皮羊膜及其浸提液体外诱导骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)向上皮细胞的分化,并探讨其机制。方法从胎儿四肢长骨分离BMSCs,扩增后采用流式细胞术分析第3代(P3)细胞表面抗原(CD29、CD34、CD71和HLA-DR)的表达,并用4,6-乙酰基-2-苯基吲哚(DAPI)标记第4代BMSCs(P4-BMSCs)。机械法去除正常胎盘羊膜上皮,制成去上皮羊膜,并制备去上皮羊膜浸提液。将DAPI标记的BMSCs接种于羊膜上,设加或不加表皮细胞生长因子(Epidermal growth factor,EGF)、类胰岛素1号生长因子(Insulin-like growth factor 1,IGF-1)、羊膜浸提液诱导组及细胞爬片对照组,体外诱导培养后,采用免疫荧光组织(细胞)化学染色学法检测各组细胞角蛋白(Cytokeratin,CK)、EGF-R和IGF-1-R的表达,并于诱导后第10天计算CK阳性细胞率。结果原代BMSCs呈典型旋涡状生长,P3细胞表达CD29和CD71,不表达CD34和HLA-DR。羊膜组和细胞爬片组BMSCs在加入EGF或IGF-1诱导后,表达EGF-R和IGF-1-R的时间较未加生长因子的对照组提前2～4 d,表达CK的时间提前2～6 d,单用羊膜组或羊膜浸提液组的表达时间差异无统计学意义(P>0.05);诱导第10天,单用羊膜或羊膜浸提液诱导组的CK阳性细胞表达率明显高于细胞爬片对照组(P<0.05);羊膜与EGF、IGF-1联合诱导组高于单用羊膜组(P<0.05);EGF诱导组高于IGF-1诱导组(P<0.05)。结论羊膜及羊膜浸提液、外源性EGF和IGF-1在体外均可诱导BMSCs向上皮细胞分化,羊膜可能主要通过其所含的细胞因子诱导BMSCs向上皮分化。     

Microwave-assisted synthesis and characterization of bioactive chitosan scaffolds doped with Au nanoparticles for mesenchymal stem cells culture

In this work we present a novel strategy for chitosan-based scaffolds. Chitosan is a versatile biopolymer obtained from waste biomass known of its favorable biological properties. Thus it can replace other polymers in the preparation of bioactive scaffolds. To increase its durability chitosan can be crosslinked into form of the hydrogel yet application of toxic crosslinkers may lead to loss of biocompability. Mesenchymal stem cells can be used in cell therapy for advanced wound treatment. However their culture requires special biomaterials application. In this article a novel microwave-assisted synthesis method for bioactive chitosan scaffolds is presented.     

Activated carbon cloth as support for mesenchymal stem cell growth and differentiation to osteocytes

Activated carbon cloth was shown to be a good support for the growth and differentiation of mesenchymal stem cells (MSCs). By using this approach, we improved the cell culture procedure and reduced the laboratory time required to obtain a very large number of MSCs suitable for utilization or differentiation.     

控释重组人骨形态发生蛋白-2和血管内皮生长因子的微囊支架对骨髓间充质干细胞向成骨细胞分化的影响

目的探讨控释重组人骨形态发生蛋白-2(recombinant huaman bone morphogenetic protein-2,rhBMP-2)及血管内皮生长因子(vascular endothelial growth factor,VEGF)微囊支架对骨髓间充质干细胞(bone marrow mesenchymal stem cells,b MSCs)向成骨细胞分化的影响。方法以聚乳酸-聚乙二醇-聚乳酸三嵌段共聚物[polylactide-poly(ethylene glycol)-polylactide,PELA]为囊材,采用复乳溶剂挥发法制备外黏附rhBMP-2内包封VEGF的微囊支架。经ELSIA法检测微囊支架在PBS中释放rhBMP-2和VEGF的浓度。将微囊支架加入bMSCs,于培养后第3、7、14天,MTT法检测微囊支架对bMSCs活性的影响,Western blot法检测微囊支架对bMSCs向成骨细胞分化过程中MAPK通路相关蛋白及碱性磷酸酶(alkaline phosphatase,ALP)表达水平的影响。结果微囊支架于PBS中培养第2天rhBMP-2释放约60%,VEGF释放约32%。随着培养时间的延长,微囊支架对bMSCs的细胞活性无明显影响(P0. 05);培养后第14天磷酸化ERK1/2及ALP表达水平均显著高于第3和7天(P 0. 05),培养后第7天显著高于第3天(P 0. 05);培养后第3、7、14天磷酸化JNK及磷酸化p38表达水平变化差异无统计学意义(P 0. 05)。结论控释rhBMP-2及VEGF的微囊支架可诱导bMSCs向成骨细胞分化,可能是通过激活MAPK通路发挥作用。     

Enhanced chondrogenic differentiation of human bone marrow mesenchymal stem cells on PCL/PLGA electrospun with different alignments and compositions

The simultaneous effect of electrospun scaffold alignment and polymer composition on chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSC) is investigated. Aligned and randomly oriented polycaprolactone/poly(lactic-co-glycolic acid) (PLGA) hybrid electrospun scaffolds with two different ratios are fabricated by electrospinning. It is found that aligned nanofibrous scaffolds support higher chondrogenic differentiation of hBMMSCs compared to random ones. The aligned scaffolds show a higher expression level of chondrogenic markers such as type II collagen and aggrecan. It is concluded that the aligned nanofibrous scaffold with higher PLGA ratio could significantly enhance hBMMSC proliferation and differentiation to chondrocytes.