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.     

人肝细胞生长因子基因真核表达质粒的构建及其在人骨髓间充质干细胞中的表达

目的构建人肝细胞生长因子(Human hepatocyte growth factor,hHGF)真核表达质粒,并检测其在人骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)中的表达及其对细胞生长的影响。方法采用密度梯度法从人骨髓中分离BMSCs,流式细胞术检测细胞表型,成脂及成骨诱导其分化。PCR扩增hHGF基因,定向克隆至pEGFP-N1载体中,构建重组表达质粒pEGFP-N1-hHGF,通过电穿孔法转染BMSCs,荧光显微镜下观察增强型绿色荧光蛋白的表达,RT-PCR及Western blot法检测hHGF基因mRNA的转录及蛋白的表达,MTT法检测hHGF对BMSCs增殖活力的影响。结果 BMSCs高表达CD29和CD44,不表达CD34和CD45;BMSCs在体外有向成脂及成骨细胞诱导分化的能力。酶切及基因测序证实,重组表达质粒pEGFP-N1-hHGF构建正确;转染后48 h观察到转染细胞中有绿色荧光蛋白表达;RT-PCR法和Western blot检测到hHGF基因在BMSCs中表达;转染hHGF基因的BMSCs增殖活力明显高于空白对照组和空载体转染组(P<0.05)。结论成功构建了hHGF基因真核表达质粒,转染人BMSCs后获得表达,表达的hHGF可促进BMSCs增殖。     

Robocast zirconia-toughened alumina scaffolds: Processing,structural characterisation and interaction with human primary osteoblasts

Zirconia-toughened alumina (ZTA) is the gold-standard ceramic in hip arthroplasty, but still lacks direct osseointegration and a metal shell, often coated with a bioactive layer, is currently required. The latter could potentially be replaced by a thinner, architectured ZTA layer, thereby allowing for larger acetabular components, with larger range of motion and lower dislocation risk. Robocasting may be an adequate technique to fabricate the architectured layer. Therefore, as a first step, this study aimed to produce ZTA scaffolds (3D-ZTA) by robocasting and assess their in vitro response. Shape retention was achieved by using a stable, well-dispersed, high solid loading ink injected in acid pH waterbath. 3D-ZTA exhibit regularly spaced microporous, rough struts and fully interconnected macroporosity. Human primary osteoblasts were homogenously distributed inside 3D-ZTA and showed increased osteogenic marker expression compared to 2D-ZTA control. Further work will focus on optimizing scaffold design to improve cell retention and extracellular matrix maturation.     

Role of iron on physical and mechanical properties of brushite cements,and interaction with human dental pulp stem cells

Improving the physical, mechanical and biological properties of brushite cements (BrC) is of a great interest for using them in bone and dental tissue engineering applications. The objective of this study was to incorporate iron (Fe) at different concentrations (0.25, 0.50, and 1.00 wt%) to BrC and study the role of Fe on phase composition, setting time, compressive strength, and interaction with human dental pulp stem cells (hDPSCs). Results showed that increase in Fe concentration increases the β-tricalcium phosphate (β-TCP)/dicalcium phosphate dihydrate (DCPD) ratio and prolongs the initial and final setting time due to effective role of Fe on stabilizing the β-TCP crystal structure and retarding its dissolution kinetic, in a dose dependent manner where the highest setting time was recorded for 1.00 wt% Fe–BrC sample. Addition of low concentrations of Fe (0.25 and 0.50 wt%) did not have adverse effect on compressive strength and strength was in the range of 5.7–7.05 (±~1.4) MPa; however, presence of 1.00 wt% Fe decreases the strength of BrC from 7.05 ± 1.57 MPa to 3.12 ± 1.06 MPa. Interaction between the BrCs and hDPSCs was evaluated by cell proliferation assay, scanning electron microscopy, and live/dead staining. Low concentrations of 0.25, and 0.50 wt% of Fe did not have any adverse effect on cell attachment and proliferation; while significant decrease in cellular activity was evident in BrC samples doped with 1.00 wt %. Together, these data show that low concentrations of Fe (equal or less than 0.50 wt %) can be safely added to BrC without any adverse effect on physical, mechanical and biological properties in presence of hDPSCs.     

A magnetic resonance-compatible perfusion bioreactor system for three-dimensional human mesenchymal stem cell construct development

Human mesenchymal stem cells (hMSCs) have significant potential for therapeutic tissue regeneration and repair. The creation of functional 3D constructs from hMSCs depends on the innate ability of MSCs to proliferate and differentiate, and is strongly influenced by the culture conditions. An inherent challenge in investigating 3D cellular construct development is the dynamic monitoring of the cellular and physiological environment over the course of construct formation. In this project, a novel 3D MR-compatible perfusion bioreactor using 3D poly(ethylene terephthalate) scaffolds was developed to provide such monitoring. The bioreactor system integrates cell seeding and growth, supports high density 3D tissue construct growth and facilitates repeated nuclear magnetic resonance (MR) signal acquisitions under both static and perfusion conditions. The reactor system also has the capacity to modulate macroscopic flow modes that simulates various tissue growth environments with repeated MR signal acquisition, providing the ability to gain insight into the dynamic interplay between the stem cells in the developing constructs and their microenvironment. Using ¹H MR spectroscopy and MR imaging, localized spectroscopic data as well as imaging-based T₂ and diffusion quantification were acquired from the hMSC growth construct for up to 40 days.     

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。     

How bone marrow-derived human mesenchymal stem cells respond to poorly crystalline apatite coated orthopedic and dental titanium implants

Due to the delayed and weak bone-implant integration in dental and orthopedic devices, there have been several attempts to enhance implant–bone interactions for rapid osseointegration. In this paper, the interactions of human bone marrow-derived stromal (mesenchymal) stem cells (hMSCs) with uncoated and coated titanium alloy implants with poorly crystalline apatite are studied. First the configuration and chemical composition of the apatite coatings and their deposition progress in different experimental conditions are investigated and discussed. Then, hMSCs are cultured on different substrates and cell attachment and proliferation are monitored and evaluated for different time intervals. Although the uncoated and coated substrates indicate good cell attachment, the differences in proliferation and morphology of the cells spread over the coated samples are significant. It is concluded that the coated samples improve the capability for accepting the cells in three-dimensional and slender shapes. The migration of hMSCs on both substrates are discussed. As such cell migration is directly associated to the osteoconduction, the findings confirm the hypothesis of enhancement in bone formation on the surface of biomimetically poorly crystalline apatite coated titanium implants. This in vitro study demonstrates that the coated samples are nontoxic and biocompatible enough for ongoing osteogenic studies in bone or dental defects in animal models in vivo.     

Chemical stiffening of constructs between polymeric microparticles based on a hyaluronic acid derivative and mesenchymal stem cells: rheological and in vitro viability studies

Our research group has recently developed microparticles of a hyaluronic acid derivative used for bottom‐up growth of microparticles/human mesenchymal stem cells (hMSCs). In this work, we investigated a strategy to increase the stiffening of aggregated constructs between microparticles and hMSCs. In particular, we applied a Michael‐type crosslinking procedure between microparticles to allow a chemically driven and cell‐compatible stiffening of constructs. Two batches of microparticles were functionalized with thiol and maleimide groups, respectively, and were then mixed to allow chemical crosslinking. The adhesion of hMSCs was controlled through addition of the adhesive peptide cyclo(‐Arg‐Gly‐Asp‐D‐Phe‐Cys) (cyRGDC). Rheological measurements performed in this study showed that the chemical stiffening strategy allows the G′ modulus of bottom‐up growing constructs to be increased, while viability tests suggest that the chemical procedure did not negatively affect cell viability compared with constructs obtained without chemical crosslinking. © 2018 Society of Chemical Industry     

An oligodeoxynucleotide that induces differentiation of bone marrow mesenchymal stem cells to osteoblasts in vitro and reduces alveolar bone loss in rats with periodontitis

To investigate the effect of oligodeoxynucleotides (ODNs) on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts, in order to find a candidate ODN with potential for the treatment of periodontitis, a series of ODNs were designed and selected to test their effect on the promotion of the differentiation of BMSCs to osteoblasts in vitro and on the repair of periodontal tissue in rats with periodontitis. It was found that MT01, one of the ODNs with the sequences of human mitochondrial DNA, stimulated the proliferation of BMSCs, the differentiation of BMSCs to osteoblasts and mRNA expression of bone-associated factors including Runx2, Osterix, OPG, RANKL and collagen I in vitro. In vivo study showed that MT01 prevented the loss of alveolar bone in the rats with periodontitis and induced the production of proteins of OPG and Osterix in the bone tissue. These results indicated that MT01 could induce differentiation of BMSCs to osteoblasts and inhibit the alveolar bone absorption in rats with periodontitis.     

Fluorescent magnetic nanoparticle-labeled mesenchymal stem cells for targeted imaging and hyperthermia therapy of in vivo gastric cancer

How to find early gastric cancer cells in vivo is a great challenge for the diagnosis and therapy of gastric cancer. This study is aimed at investigating the feasibility of using fluorescent magnetic nanoparticle (FMNP)-labeled mesenchymal stem cells (MSCs) to realize targeted imaging and hyperthermia therapy of in vivo gastric cancer. The primary cultured mouse marrow MSCs were labeled with amino-modified FMNPs then intravenously injected into mouse model with subcutaneous gastric tumor, and then, the in vivo distribution of FMNP-labeled MSCs was observed by using fluorescence imaging system and magnetic resonance imaging system. After FMNP-labeled MSCs arrived in local tumor tissues, subcutaneous tumor tissues in nude mice were treated under external alternating magnetic field. The possible mechanism of MSCs targeting gastric cancer was investigated by using a micro-multiwell chemotaxis chamber assay. Results show that MSCs were labeled with FMNPs efficiently and kept stable fluorescent signal and magnetic properties within 14 days, FMNP-labeled MSCs could target and image in vivo gastric cancer cells after being intravenously injected for 14 days, FMNP-labeled MSCs could significantly inhibit the growth of in vivo gastric cancer because of hyperthermia effects, and CCL19/CCR7 and CXCL12/CXCR4 axis loops may play key roles in the targeting of MSCs to in vivo gastric cancer. In conclusion, FMNP-labeled MSCs could target in vivo gastric cancer cells and have great potential in applications such as imaging, diagnosis, and hyperthermia therapy of early gastric cancer in the near future.     

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.     

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.     

De-painting with high-speed water jets: Paint removal process and substrate surface roughness

Although the use of water jets for paint removal processes is an accepted procedure, there are just a few studies known which discuss parameter optimization and surface topography in some detail. The paper investigates the effects of water jet kinetic energy and stand-off distance on the mass loss of an organic paint system applied to a steel substrate. It was shown that the material removal process was characterized by a combination of loading intensity and loading frequency. Water drops, formed in the water jet at long stand-off distances, played a notable role. For rather high water jet energies, mass loss exhibited high values at high stand-off distances. For lower water jet energies, however, maximum material loss values appeared at a critical stand-off distance. The transition water jet energy was 600 kJ. It could be shown that the steel substrate topography was not compromised due to secondary surface preparation by water jets. Secondary blast cleaning, however, reduced the profile of the substrate.     

Chitosan-gelatin/hydroxyapatite-based scaffold associated with mesenchymal stem cells differentiate into osteoblasts improves the surface of the bone lesion in mice C57BL/6J

Extensive injuries to bone tissue are still considered a significant clinical challenge; therefore, developing new bone tissue engineering (BTE) strategies is still necessary. This work aims to construct and characterize a chitosan-gelatin/hydroxyapatite-based (CG/H) scaffold to provide well-design support for mesenchymal stem cell (MSC) growth and differentiation to osteoblasts. First, the CG/H scaffolds are construct by freeze-drying. Then, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, x-ray diffraction, water uptake, and degradation profiles evaluate the material's surface. In addition, the CG/H morphological, biochemical, and MSC adhesion processes and growth behavior are also assess, indicating reasonable adhesion rates to the surface, low material cytotoxicity, and excellent alkaline phosphatase activity compared to control on the cellular framework. Based on these results, we obtain a highly biocompatible scaffold and that can support osteoblast differentiation. Finally, the in vivo studies demonstrate the CG/H scaffold with MSC adhere is capable of differentiating into osteoblasts, and the application of this scaffold is able to significantly enhance the closure of the bone lesion. Therefore, the CG/H scaffold has potential clinical application for bone regeneration.     

一种分离培养人脐带Wharton's jelly间充质干细胞的新方法

目的探讨一种分离培养人脐带Wharton′s jelly间充质干细胞(mesenchymal stem cell,MSC)的新方法。方法取人脐带组织,除去动静脉后剪碎成2～5 mm3,将组织碎片浸入4 g/LⅠ型胶原酶和1 g/L透明质酸酶的混合液中,于37℃处理1 h,再用0.25%胰蛋白酶在相同条件下消化30 min,得到的消化液经70μm细胞滤网过滤后,制备单个细胞悬液,培养并传代。取P1、P3、P7代脐带Wharton′s jelly MSC,绘制细胞生长曲线;取P3代细胞,采用流式细胞术检测细胞表面标记分子,并分别采用成骨和成脂诱导培养基进行成骨及成脂诱导分化,茜素红染色和油红O染色观察结果。结果 P1、P3代脐带Wharton′s jelly MSC的增殖能力强,且P1代细胞的增殖能力强于P3代,P7代细胞的增殖能力较P3代细胞有所减弱。P3代脐带Wharton′s jelly MSC高表达CD90(99.8%)、CD105(100%)和CD166(100%),低表达CD45(0.3%)、CD14(0.1%)、CD34(0.2%)和CD79a(0.3%),不表达HLA-DR。P3代Wharton′s jellyMSC经成骨诱导后,茜素红染色可见红色结节;经成脂诱导后,油红O染色可见脂质沉积。结论本方法获得的Wharton′s jelly MSC活性好,增殖能力强,为后续实验研究及临床应用提供了理想的种子细胞。     

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.     

Calcium silicate/calcium aluminate composite biocement for bone restorative application: synthesis,characterisation and in vitro biocompatibility

Pure β-dicalcium silicate and monocalcium aluminate powder were prepared by Pechini method. A series of calcium silicate/calcium aluminate cements (CSC/CAC) were prepared. The setting time, crystalline phases, microstructures, compressive strength, cells attachment and silicon release of the cements were investigated. The results indicate that the setting time of CSC/CAC was shorter than that of either CSC or CAC. The hydration products in CSC/CAC composite are gehlenite (Ca₂Al₂SiO₇·8H₂O), calcium aluminate hydrate (Ca₃Al₂O_6?×?H₂O), and katoite (Ca₂Al₂O₆·6H₂O). Platelike crystals were found in the microstructure. The liquid to powder ratio has a significant effect on the porosity and the strength of CSC/CAC. The MC3T3 cells attached well to the surfaces of CSC/CAC. However, the cells proliferation on the surface of 7S3A was better than that of 3S7A due to its higher silicon release. In general, CSC/CAC exhibits good biocompatibility and relative high strength, and may be suitable for some non-load bearing bone restorative applications.     

Investigation of biocompatible nanosized materials for development of strong calcium phosphate bone cement: Comparison of nano-titania,nano-silicon carbide and amorphous nano-silica

The present paper deals with the effect of adding SiC, TiO₂ and SiO₂ nanoparticles on setting time, mechanical strength and hydraulic reactions of calcium phosphate cements (CPCs). The initial and final setting times of CPC increased by adding both nano-SiC and nano-TiO₂ additives but decreased by using nano-silica. Nano-titania and nano-silica had great effect on compressive strength of as-set CPC whereas slight changes were found by using nano-SiC. Although a sharp increase in compressive strength of all cements was observed by soaking them in physiological solution, the soaked additive-free cements and nano-SiO₂-added ones exhibited the greatest strength values. The results showed that adding these nano-additives did not influence on conversion rate of cement reactants to apatite phase during soaking in physiological solution period but the morphology of the formed phase was almost different. Overall, the results determined that nano-SiO₂ and nano-TiO₂ particles were appropriate additives to improve short-term mechanical strength of CPCs a(s-set CPCs), though nano-SiO₂ was found more effective because it improves the long-term mechanical strength of CPC (after soaking) too.