Viability Of Human Mesenchymal Stem Cells Research Articles

Nanoarchitectonics of Bactericidal Coatings Based on CaCO3-Nanosilver Hybrids.

Antimicrobial coatings provide protection against microbes colonization on surfaces. This can prevent the stabilization and proliferation of microorganisms. The ever-increasing levels of microbial resistance to antimicrobials are urging the development of alternative types of compounds that are potent across broad spectra of microorganisms and target different pathways. This will help to slow down the development of resistance and ideally halt it. The development of composite antimicrobial coatings (CACs) that can host and protect various antimicrobial agents and release them on demand is an approach to address this urgent need. In this work, new CACs based on microsized hybrids of calcium carbonate (CaCO3) and silver nanoparticles (AgNPs) were designed using a drop-casting technique. Polyvinylpyrrolidone and mucin were used as additives. The CaCO3/AgNPs hybrids contributed to endowing colloidal stability to the AgNPs and controlling their release, thereby ensuring the antibacterial activity of the coatings. Moreover, the additives PVP and mucin served as a matrix to (i) control the distribution of the hybrids, (ii) ensure mechanical integrity, and (iii) prevent the undesired release of AgNPs. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) techniques were used to characterize the 15 μm thick CAC. The antibacterial activity was determined against Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa, three bacteria responsible for many healthcare infections. Antibacterial performance of the hybrids was demonstrated at concentrations between 15 and 30 μg/cm2. Unloaded CaCO3 also presented bactericidal properties against MRSA. In vitro cytotoxicity tests demonstrated that the hybrids at bactericidal concentrations did not affect human dermal fibroblasts and human mesenchymal stem cell viability. In conclusion, this work presents a simple approach for the design and testing of advanced multicomponent and functional antimicrobial coatings that can protect active agents and release them on demand.

Adherence and activation of human mesenchymal stromal cells on brushite coated porous titanium

Tri-calcium-phosphate Ca3(PO4)2 is considered a promising material to improve implant and bone tissue linking. Titanium (Ti), a highly porous material, was coated with crystalline brushite using an electrochemical process so that it could be used as an implant material. The aim of this research is to get a faster ingrowth of bone-tissue inside the porous structure. The crystalline coating was characterized by the standard XRD and SEM techniques. Additionally, the phase evolution of Ca3(PO4)2-crystals with different kinds of sterilization techniques was observed. In-vitro tests on the biocompatibility of the coating were done with human mesenchymal stem cells (HMSCs). The HMSCs are the most promising cell type for regenerative medicine and tissue engineering owing to their ability to differentiate into several tissues, such as, bone, cartilage, tendon, and muscle. For the treatment of local bone defects expanded HMSCs could be loaded on Ca3(PO4)2 coated metallic matrices and delivered to the target site. A possible application is brushite-coated porous titanium as a carrier matrix. To determine whether cell functions of HMSCs are influenced by the brushite surface, HMSCs were cultured on brushite-coated and uncoated porous titanium specimens. The viability and proliferation of HMSCs were successfully analyzed. Additionally, the release of cytokines (IL-6, IL-8, IL-11) were quantified as a marker of cell activation. The viable cells could be detected on specimens after 48 h initial seeding. After 8 days cell number on both specimens increased, although the proliferation on brushite-coated titanium was decreased compared to the uncoated surfaces. The release of both, IL-8 and IL-11 increased significantly due to the cultivation of HMSCs on the coated specimens for 8 days.

Hydroxyapatite nanoparticle-modified porous bone grafts with improved cell attachment.

Emulsion-templated foams have displayed promise as injectable bone grafts; however, the use of a surfactant as an emulsifier resulted in relatively small pores and impedes cell attachment. Hydroxyapatite nanoparticles were explored as an alternative stabilizer to address these limitations. To this end, hydroxyapatite nanoparticles were first modified with myristic acid to generate the appropriate balance of hydrophobicity to stabilize a water-in-oil emulsion of neopentyl glycol diacrylate and 1,4-butanedithiol. In situ surface modification of the resulting foam with hydroxyapatite was confirmed with elemental mapping and transmission electron microscopy. Nanoparticle-stabilized foams displayed improved human mesenchymal stem cell viability (91 ± 5%) over surfactant-stabilized foams (23 ± 11%). Although the pore size was appropriate for bone grafting applications (115 ± 71 μm), the foams lacked the interconnected architecture necessary for cell infiltration. We hypothesized that a co-stabilization approach with both surfactant and nanoparticles could be used to achieve interconnected pores while maintaining improved cell attachment and larger pore sizes. A range of hydroxyapatite nanoparticle and surfactant concentrations were investigated to determine the effects on microarchitecture and cell behavior. By balancing these interactions, a co-stabilized foam was identified that possessed large, interconnected pores (108 ± 67 μm) and improved cell viability and attachment. The co-stabilized foam was then evaluated as an injectable bone graft including network formation, microscale integration with bone, push out strength, and compressive properties. Overall, this work demonstrated that in situ surface modification with nHA improved cell attachment while retaining desirable bone grafting features and injectability.

In the presence of TGF-\u03b21, Asperosaponin VI promotes human mesenchymal stem cell differentiation into nucleus pulposus like- cells

BackgroundThe regeneration of nucleus pulposus (NP) cells is an effective method to prevent intervertebral disc degeneration (IVDD). In this study, we investigated the role of Asperosaponin VI (ASA VI), isolated from a traditional Chinese medicine (TCM), the root of Dipsacus asper Wall, in promoting human mesenchymal stem cell (HMSC) proliferation and differentiation into NP-like cells and explored the possible mechanism of action.MethodsThe effects of ASA VI on HMSC viability and proliferation were determined by the XTT method and EDU staining. Then, Real-time qPCR, immunocytochemistry and immunofluorescence assays were used to measure the effect of ASA VI on the expression of extracellular matrix (ECM) components, such as COL2A1, aggrecan, SOX9, KRT19, PAX1, and glycosaminoglycans (GAGs), in NP cells. In addition, Western blot assay was used to measure the expression of p-ERK1/2 and p-smad2/3.ResultsASA VI was able to promote the proliferation and differentiation of HMSCs into NP-like cells, and the optimum concentration was 1 mg/L. Western blot assay indicated that the possible mechanism might be related to the activation of p-ERK1 / 2 and p-Smad2 / 3.ConclusionsASA VI can promote the proliferation and differentiation of HMSCs into NP-like cells, which can potentially be used as a treatment for IVDD.

Synthesis and cytocompatibility analysis of carbon nanodots derived from palmyra palm leaf for multicolor imaging applications

A humble and environmental benign preparation of carbon nanodots (CDs) utilizing palmyra leaves as a sustainable precursor, by hydrothermal method, is reported. The morphology, structure, optical and thermal behavior of CDs was investigated using transmission electron microscopy, photoluminescence spectroscopy UV–Vis spectroscopy, X-ray diffractometer, Fourier transform infrared spectroscopy and thermogravimetric analysis. The fluorescent CDs have a spherical shape and size of around 5–10 nm. The TEM images suggest that crystallinity of the carbon nanodots with a lattice parameter of 0.238 nm, corresponding to the (100) diffraction facets of graphite. The synthesized CDs biocompatibility was assessed using the cell viability assay and acridine orange/ethidium bromide (AO/EB) dual staining in human mesenchymal stem cells (hMSCs). The cell viability assay results exhibit that CDs slightly decrease the human mesenchymal stem cells viability at high dose. However, no remarkable changes were observed in cell viability. Furthermore, we assessed the CDs influence on nuclear morphology of hMSCs. These results clearly suggest that CDs treated cells have healthy and intact nuclei. Overall, our study results revealed that CDs have excellent compatibility on cells. Thus, the synthesized CDs can be used as versatile fluorescent probe for cellular imaging because of their excitation-dependent emission. Furthermore, we utilize the CDs as fluorescent probe for cellular imaging using confocal microscope, resulting tunable fluorescent emission. The CDs can enter the cells and accumulate in cytoplasmic region, symptomatic of congenial potential for multicolor imaging.

In vitro Microglia Cell Line and Human Mesenchymal Stem Cells Viability IS Inhibited by Hydrogen Gas (P1.2-093)

In vitro Microglia Cell Line and Human Mesenchymal Stem Cells Viability IS Inhibited by Hydrogen Gas (P1.2-093)

Evaluating Nonintegrating Lentiviruses as Safe Vectors for Noninvasive Reporter-Based Molecular Imaging of Multipotent Mesenchymal Stem Cells.

Reporter gene-based molecular imaging can provide invaluable information on the fate of cellular therapies postimplantation. Integrating lentiviral vectors (ILVs) are commonly used for stably engineering cells; however, their potential for insertional mutagenesis poses a significant safety concern and barrier to widespread clinical adoption. In cells that slowly divide or are postmitotic in vivo, such as mesenchymal stem cells (MSCs), nonintegrating lentiviral vectors (NILVs) may be a safer alternative option because NILVs remain episomal and can provide prolonged expression profiles. Here, NILVs coexpressing fluorescence and bioluminescence imaging (BLI) reporters were engineered and used to longitudinally image the viability of human MSCs in vitro and in vivo in mice. In vitro, ILV-transduced cancer cells and MSCs maintained steady reporter gene expression over time, whereas NILV-transduced cells progressively lost signal. NILV reporter loss was accelerated in highly proliferative cancer cells compared with less proliferative MSCs. In vivo, ILV- and NILV-transduced MSCs were each detectable with BLI postintramuscular implantation, with significantly higher ILV-based signal compared with NILV-based signal. BLI signal was observed to similarly diminish over time for both cell populations, which was attributed to cell death. Despite the reduced signal intensity with NILVs and the minimal number of cells injected (40,000), live NILV-transduced MSCs were reliably visualized for up to 2 weeks. Safety is a concern for future clinical reporter gene applications. We present NILVs as a safe means of imaging reporter gene expression for slowly dividing or nondividing cells and showed effective tracking of the viability of a small number of live transplanted MSCs over time with optical imaging. Future work will evaluate improvements to episomal NILV reporter expression, explore sensitive clinically relevant reporters, and apply this approach to clinically relevant MSC applications in preclinical models. NILVs may have broad clinical applications for expression of imaging reporters and other gene products in MSC-based therapies.

Phenotypic and Genetic Evaluation of the Influence of Pseudomonas aeruginosa Culture Fractions on the Human Mesenchymal Stem Cells Viability, Apoptotic Pathways and Cytokine Profile.

The objective of this study was to investigate the effects of P. aeruginosa PAO1 cellular and soluble culture fractions on human mesenchymal stem cells (MSCs) death signaling pathways and cytokine profile. The bone marrow isolated MSCs, incubated for different periods of time with one of the three P. aeruginosa PAO1 culture fractions, i.e. low density whole cultures, heat inactivated bacterial cultures sediments and sterile supernatants, were submitted to the following assays: i) fluorescence microscopy evaluation of cellular morphology and viability; ii) bax, caspase 9, relA and bcl-2 genes expression analysis by qRT-PCR; and iii) quantification of the level of IL-1β, IL-6, IL-8 and IL-10 cytokines released in the MSCs supernatants determined by ELISA. Results were statistically analyzed using the GraphPad In Stat software. The PAO1 whole cultures exhibited the most relevant influences, impacting on MSCs morphology and viability, interfering with apoptotic pathways and significantly stimulating the production of IL-1β and IL-10, while decreasing the production of IL-6 and IL-8. The culture supernatants increased the production of IL-1β and reduced the secretion of all other tested cytokines, while heat-inactivated bacterial cells significantly stimulated both IL-1β and IL-10 production. These data could suggest that in vivo, the fate of P. aeruginosa infection depends on the proportion between different bacterial culture fractions (i.e. the number of viable bacterial cells, the number of dead cells and the amount of bacterial soluble products accumulated locally) that could be influenced by the initial infective dose, by the host defense mechanisms, and also by the administered antimicrobial treatment that may thus interfere with the evolution and magnitude of the induced lesions.

Osteoinductive PolyHIPE Foams as Injectable Bone Grafts.

We have recently fabricated biodegradable polyHIPEs as injectable bone grafts and characterized the mechanical properties, pore architecture, and cure rates. In this study, calcium phosphate nanoparticles and demineralized bone matrix (DBM) particles were incorporated into injectable polyHIPE foams to promote osteoblastic differentiation of mesenchymal stem cells (MSCs). Upon incorporation of each type of particle, stable monoliths were formed with compressive properties comparable to control polyHIPEs. Pore size quantification indicated a negligible effect of all particles on emulsion stability and resulting pore architecture. Alizarin red calcium staining illustrated the incorporation of calcium phosphate particles at the pore surface, while picrosirius red collagen staining illustrated collagen-rich DBM particles within the monoliths. Osteoinductive particles had a negligible effect on the compressive modulus (∼30 MPa), which remained comparable to human cancellous bone values. All polyHIPE compositions promoted human MSC viability (∼90%) through 2 weeks. Furthermore, gene expression analysis indicated the ability of all polyHIPE compositions to promote osteogenic differentiation through the upregulation of bone-specific markers compared to a time zero control. These findings illustrate the potential for these osteoinductive polyHIPEs to promote osteogenesis and validate future in vivo evaluation. Overall, this work demonstrates the ability to incorporate a range of bioactive components into propylene fumarate dimethacrylate-based injectable polyHIPEs to increase cellular interactions and direct specific behavior without compromising scaffold architecture and resulting properties for various tissue engineering applications.

Is cell viability always directly related to corrosion resistance of stainless steels?

It has been frequently reported that cell viability on stainless steels is improved by increasing their corrosion resistance. The question that arises is whether human cell viability is always directly related to corrosion resistance in these biostable alloys. In this work, the microstructure and in vitro corrosion behavior of a new class of medical-grade stainless steels were correlated with adult human mesenchymal stem cell viability. The samples were produced by a powder metallurgy route, consisting of mechanical alloying and liquid-phase sintering with a sintering aid of a eutectic Mn–Si alloy at 1050°C for 30 and 60min, leading to nanostructures. In accordance with transmission electron microscopic studies, the additive particles for the sintering time of 30min were not completely melted. Electrochemical impedance spectroscopic experiments suggested the higher corrosion resistance for the sample sintered for 60min; however, a better cell viability on the surface of the less corrosion-resistant sample was unexpectedly found. This behavior is explained by considering the higher ion release rate of the Mn–Si additive material, as preferred sites to corrosion attack based on scanning electron microscopic observations, which is advantageous to the cells in vitro. In conclusion, cell viability is not always directly related to corrosion resistance in stainless steels. Typically, the introduction of biodegradable and biocompatible phases to biostable alloys, which are conventionally anticipated to be corrosion-resistant, can be advantageous to human cell responses similar to biodegradable metals.

Poly(lactic-co-glycolic acid)/gelatin electrospun scaffolds seeded with human mesenchymal stem cells for skin tissue engineering

Event Abstract Back to Event Poly(lactic-co-glycolic acid)/gelatin electrospun scaffolds seeded with human mesenchymal stem cells for skin tissue engineering Nadia Vázquez1, Casandra Chaires1, Beatriz Hernández1, Alfredo Maciel2, Ricardo Vera-Graziano2, Mathieu Hautefeuille3, Miguel A. Herrera1 and Andrés Castell1 1 Universidad Nacional Autónoma de México, Facultad de Medicina, Mexico 2 Universidad Nacional Autónoma de México, Instituto de Investigaciones en Materiales, Mexico 3 Universidad Nacional Autónoma de México, Facultad de Ciencias, Mexico Introduction: Treatment options for burns are expensive and inaccessible to most people, so finding cheap to manufacture scaffolding materials is a priority. Materials of scaffolds must to promote adhesion, proliferation and cellular differentiation and finally, the generation of new tissues[1]. Polylactic-co-glycolic acid (PLGA) and gelatin (GE) are materials that posses biocompatibility and biodegradability. Therefore, the aim of this study was to evaluate cell viability and biocompatibility of a PLGA/Ge scaffold seeded with human mesenchymal stem cells (HMSC) and implanted subcutaneously in mice. Materials and Methods: PLGA 18% (w/v) solution (LA/GA 50:50, Mw=54,000) and GE 10% (w/v) solution (from bovine skin) were prepared separately by dissolving both in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP) as solvent. Fibers were obtained from PLGA/GE 9:1 solution. Scaffolds were fabricated by electrospinning (flow rate: 0.3ml/hr, voltage: 10Kv and distance: 10cm). The hydrophobic was measured by contact angle method and the morphology was evaluated by SEM. HMSC´s were isolated from Warthon jelly and stained with fluorescent cell tracker before were seeded on scaffolds. Viability cell were evaluated through LIVE/DEAD® Cell Viability Assays. After 5 days of culture, scaffolds containing HMSC´s were implanted subcutaneously in Balb/c mice. Skin samples were obtained at 5, 10, 15 and 30 days. An immunofluorescent analysis and H&E staining were made. Results and Discussion: Scaffold fibers had an average thickness of 0.6nm and a hydrophilic surface with a contact angle of 39°. HMSC's seeded on scaffolds showed a viability of 95%. Due to the low porosity of the scaffolds (5um), the cells were unable to migrate into the network, but remained viable with high confluence in the area where they were seeded. When scaffolds in murine skin were analyzed it was observed that HMSC’s were viable and mouse fibroblasts colonized the scaffold. Granuloma lesions and epitheliod cells were observed, but Langhans cells were present. The data shown indicated that PLGA/GE[1] scaffolds allowed cell viability in vitro and in vivo and promoted the migration of fibroblasts. Conclusion: PLGA/GE scaffolds showed to maintained viability and confluence cellular of HMSC´s seeded on them during several days. Furthermore, scaffolds were biocompatible when were implanted in mice, so they can have potential utility as wound dressing in skin lesions. PAEP grant

Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature.

The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO2 (scCO2) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO2 treatment effectively inactivates microorganisms including bacterial spores. We established a scCO2 sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO2 sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO2 treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO2 treatment of these materials and scaffolds. We conclude that scCO2 sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.

Human mesenchymal stem cell viability, proliferation and differentiation potential in response to ceramic chemistry and surface roughness

We investigated the effect of the ceramic chemistry and surface roughness of pure α-tricalcium phosphate, and also αTCP doped with either 1.5wt% or 3.0wt% of dicalcium silicate (C2S), on the response of adult human mesenchymal stem cells (ahMSCs). AhMSCs were plated onto ceramic discs, prepared by a solid-state reaction. After being sintered, some samples were polished up to 1μm, while others were kept as manufactured, which resulted in two surface roughness grades. Viability, proliferation and osteoinductive capacity were determined following various incubation periods.The results showed a non-cytotoxic effect after an indirect apoptosis test. Cell adhesion and proliferation were surface roughness-sensitive and increased proportionally to the roughness of materials. These observations became more evident in the unpolished αTCP ceramic doped with 1.5wt% C2S, which induced osteoblastic differentiation as a result of the roughness and increased concentration of the C2S solid solution in αTCP.

Galactose grafting on poly(ε-caprolactone) substrates for tissue engineering: a preliminary study

The grafting of galactose units onto poly(ε-caprolactone) (PCL) substrates by a wet chemistry two-step procedure is proposed. Even though a reduction of hardness from 0.58–0.31GPa to 0.12–0.05GPa is achieved, the chemical functionalization does not negatively affect the tensile modulus (332.2±31.3MPa and 328.5±34.7MPa for unmodified and surface-modified PCL, respectively) and strength (15.1±1.3MPa and 14.8±1.5MPa as assessed before and after the surface modification, respectively), as well as the mechanical behaviour evaluated through small punch test. XPS and enzyme-linked lectin assay (ELLA) demonstrate the presence, and also the correct exposition of the saccharidic epitope on PCL substrates. The introduction of carbohydrate moieties on the PCL surfaces clearly enhances the hydrophilicity of the substrate, as the water contact angle decreases from 82.1±5.8° to 62.1±4.2°. Furthermore, preliminary biological analysis shows human mesenchymal stem cell viability over time and an improvement of cell adhesion and spreading.

The effect of temperature on the viability of human mesenchymal stem cells

IntroductionImpaction allograft with cement is a common technique used in revision hip surgeries for the last 20 years. However, its clinical results are inconsistent. Recent studies have shown that mesenchymal stem cells (MSCs) seeded onto allograft can enhance bone formation. This in vitro study investigates whether the increase in temperature related to the polymerisation of bone cement will affect the viability of human MSCs.MethodsThe viability of human MSCs was measured after incubating them at temperatures of 38°C, 48°C and 58°C; durations 45 seconds, 80 seconds and 150 seconds. A control group was kept at 37°C and 5% carbon dioxide for the duration of the investigation (7 days). During the course of the study the human MSCs were analysed for cell metabolic activity using the alamarBlue™ assay, cell viability using both Trypan Blue dye exclusion and calcein staining under fluorescent microscopy, and necrosis and apoptosis using Annexin V and propidium iodide for flow cytometric analysis. A one-way analysis of variance with a priori Dunnett’s test was used to indicate the differences between the treatment groups, when analysed against the control. This identified conditions with a significant difference in cell metabolic activity (alamarBlue™) and cell viability (Trypan Blue).ResultsResults showed that cell metabolism was not severely affected up to 48°C/150 seconds, while cells in the 58°C group died. Similar results were shown using Trypan Blue and calcein analysis for cell viability. No significant difference in apoptosis and necrosis of the cells was observed when human MSCs treated at 48°C/150 seconds were compared with the control group.ConclusionsThe study suggests that human MSCs seeded onto allograft can be exposed to temperatures up to 48°C for 150 seconds. Exposure to this temperature for this time period is unlikely to occur during impaction allograft surgery when cement is used. Therefore, in many situations, the addition of human MSCs to cemented impaction grafting may be carried out without detrimental effects to the cells. Furthermore, previous studies have shown that this can enhance new bone formation and repair the defects in revision situations.

The effects of Ca2SiO4–Ca3(PO4)2 ceramics on adult human mesenchymal stem cell viability, adhesion, proliferation, differentiation and function

Bioceramic samples with osteogenic properties, suitable for use in the regeneration of hard tissue, were synthesized. The materials consisting of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5wt.% or 3.0wt.% of dicalcium silicate (C2S) in the system Dicalcium Silicate–Tricalcium Phosphate (C2S–TCP) were obtained by solid state reaction. All materials were composed of a single phase, αTCP in the case of a pure material, or solid solution of C2S in αTCP (αTCPss) for the doped αTCP. Viability, proliferation and in vitro osteoinductive capacity were investigated by seeding, adult mesenchymal stem cells of human origin (ahMSCs) which were CD73+, CD90+, CD105+, CD34− and CD45− onto the 3 substrates for 30days.Results show a non-cytotoxic effect after applying an indirect apoptosis test (Annexin V/7-AAD staining), so ahMSCs adhered, spread, proliferated and produced extracellular matrix (Heparan-sulfate proteoglycan (HS) and osteopontin (OP)) on all the ceramics studied. Finally, the cells lost the cluster differentiation marker expression CD73, CD90 y CD105 characteristic of ahMSCs and they showed an osteoblastic phenotype (Alkaline phosphatase activity (ALP), Osteocalcin production (OC), Collagen type I expression (Col-I), and production of mineralization nodules on the extracellular matrix). These observations were more evident in the αTCP ceramic doped with 1.5wt.% C2S, indicating osteoblastic differentiation as a result of the increased concentration of solid solution of C2S in αTCP (αTCPss). Overall, these results suggest that the ceramics studied are cytocompatible and they are able to induce osteoblastic differentiation of undifferentiated ahMSCs.

In Reply: Emerging Approaches for Cardiovascular Stem Cell Imaging

To the Editor: We appreciate the careful and thoughtful letter by Dr. Gholamrezanezhad regarding our recent review article about stem cell labeling and tracking in cardiovascular disease [1]. Dr. Gholamrezanezhad expresses two concerns that he feels were inadequately covered in the article. The first concern is about the deleterious effects of exposure of stem cells to radiotracers. As a review article, with a limitation of 50 references, we chose to highlight the most recent findings and provide our interpretation of the evolving field over the past several decades. However, with regard to references to radiotracer toxicity in our review, we cited literature that primarily discussed radionuclide reporter gene labeling [2–6]. The topic of the toxicity of direct radiotraccer labeling of stem/progenitor cells with 111-Indium oxine (and/or 111-Indium tropolone), including the manuscript by Dr. Gholamrezanezhad and coauthors cited in his letter to the editor, was studied extensively by many authors [7–10] more than 5 years ago, and, therefore, was excluded from the review to provide space for more recent topical information. But, in order to provide a response to his concerns, I would refer him to Brenner et al. [7], who studied toxicity in hematopoietic progenitor cells (HPCs), based not only on viability but also on proliferation and differentiation assays. Those authors concluded that HPCs were more susceptible to radiation-induced toxicity than endothelial progenitor cells, which were also studied by radiolabeling previously [8]. Subsequently, our group investigated the homing capabilities of canine bone marrow–derived mesenchymal stem cells (MSCs) using 111-Indium oxine and superparamagnetic iron oxide labeling [9]. Based not only on viability, but also more rigorous metabolic and differentiation assays, we concluded that the choice of labeling dose per cell was important to maintain the pluripotency and metabolic activity of MSCs. We speculated that some of the differences seen in homing capabilities by Brenner et al. [7] and Aicher et al. [8] may be related to labeling dose, ie, toxicity. Nonetheless, in doses relevant for detection using clinical SPECT scanners, MSC labeling with 111-Indium oxine could be performed without sacrificing MSC pluripotency [9]. In an elegant paper by Jin et al. [10], the authors determined that viability was maintained for 2 weeks using a detectable labeling dose of 0.14 Bq/canine MSC. Approximately 5 years later, Gholamrezanezhad et al. have published a paper using a minimum dose >5 times the recommended dose by Jin et al., and found, not surprisingly, that these doses had a negative effect on human MSC viability. One of the tenets of cellular labeling, as we paraphrased in the review by Frangioni et al. [11], is that the label be “non-toxic, sensitive, and specific, and should permit long-term follow up.” For direct radiotracer labeling, as with ionizing radiation from X-rays, any exposure of a naive stem/progenitor cell is always a concern and should be kept to the minimum possible dose. Clearly, the toxicity to 111-Indium oxine of MSCs is different than lymphocytes and other cell types. Moreover, species differences will need to be clearly evaluated. We are, however, concerned about the assumption made by Dr. Gholamrezanezhad (in the abstract cited in his letter to the editor) that radiolabeled cells administered simultaD. A. Kedziorek :D. L. Kraitchman (*) Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University, Baltimore, MD, USA e-mail: dkraitc1@jhmi.edu

Imaging Gene Expression in Human Mesenchymal Stem Cells: From Small to Large Animals

To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning. Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 x 10(6) transduced (n = 5) and control nontransduced (n = 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphate-buffered saline (PBS) (n = 4) or matrigel (n = 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed. In cell culture, there was a viral dose-dependent increase of gene expression and FHBG accumulation in human MSCs. Human MSC viability was 96.7% (multiplicity of infection, 250). Cardiac FHBG uptake in rats was significantly elevated (P < .0001) after human MSC injection (0.0054% injected dose [ID]/g +/- 0.0007 [standard deviation]) compared with that in the remote myocardium (0.0003% ID/g +/- 0.0001). In swine, myocardial radiotracer uptake was not elevated after injection of up to 100 x 10(6) human MSCs (PBS group). In the matrigel group, signal-to-background ratio increased to 1.87 after injection of 100 x 10(6) human MSCs and positively correlated (R(2) = 0.97, P < .001) with the number of administered human MSCs. Reporter gene imaging in human MSCs can be translated to large animals. The study highlights the importance of co-administering a "scaffold" for increasing intramyocardial retention of human MSCs.

Screening for 3D Environments That Support Human Mesenchymal Stem Cell Viability Using Hydrogel Arrays

In this study we generated 3D poly(ethylene glycol) (PEG) hydrogel arrays to screen for the individual and combinatorial effects of extracellular matrix (ECM) degradability, cell adhesion ligand type, and cell adhesion ligand density on human mesenchymal stem cell (hMSC) viability. In particular, we explored the influence of two well-characterized ECM-derived cell adhesion ligands: the fibronectin-derived Arg-Gly-Asp-Ser-Pro (RGDSP) sequence, and the laminin-derived Ile-Lys-Val-Ala-Val (IKVAV) sequence. PEG network degradation, the RGDSP ligand, and the IKVAV ligand each individually increased hMSC viability in a dose-dependent manner. The RGDSP ligand also improved hMSC viability in a dose-dependent manner in degradable PEG hydrogels, while the effect of IKVAV was less pronounced in degradable hydrogels. Combinations of RGDSP and IKVAV promoted high viability of hMSCs in nondegradable PEG networks, while the combined effects of the ligands were not significant in degradable PEG hydrogels. Although hMSC spreading was not commonly observed within PEG hydrogels, we qualitatively observed hMSC spreading after 5 days only in degradable PEG hydrogels prepared with 2.5 mM of both RGDSP and IKVAV. These results suggest that the enhanced throughput approach described herein can be used to rapidly study the influence of a broad range of ECM parameters, as well as their combinations, on stem cell behavior.

The influence of the RGD peptide motif and its contextual presentation in PEG gels on human mesenchymal stem cell viability.

An investigation was undertaken into the method of delivery of RGD peptide motifs to adult human mesenchymal stem cells (hMSCs) encapsulated in poly(ethylene glycol) (PEG) hydrogel systems. Previous studies have shown that the viability of hMSCs encapsulated in bio-inert hydrogels, such as PEG-based gels, decreases over time. hMSCs are an adhesive-dependent cell type, requiring attachment sites to maintain their survival and function. The incorporation of tethered RGD peptide motifs was found to sustain a high level of hMSC survival in PEG gels. However, previous reports are largely limited to pendantly tethered RGD in gels; therefore, further investigation into hMSCs' affinity for and response to the contextual presentation of RGD was studied using varying methods of RGD attachment to the PEG gel, as well as delivery of soluble RGD peptides. Studies with encapsulated hMSCs showed that the constrained RGD peptide, bound via two links to the PEG gel, promoted approximately 60% cell survival, while tethering the RGD as a pendant group, bound via a single link to the PEG gel, promoted approximately 80% cell survival. Interestingly, incorporating a glycine spacer arm to the RGD pendant tether further enhanced survival to approximately 88%. Investigations with solubly delivered peptides resulted in a dramatic decrease in cell viability with time, eventually leading to survival that was similar to that of unmodified PEG gels. Integrin staining for alphavbeta3 and alpha4, as well as focal adhesion staining, correlates to the trends in hMSC survival for covalently-bound RGD motifs and a loss of viability for the solubly delivered peptide systems.