Biological performance of novel phosphate-based glass microspheres for mesenchymal stem cell therapy in osteoporotic patients

In this study, degradable phosphate-based bulk or porous glass microspheres (BGMS or PGMS), with nominal molar compositions of P45-(45P2O5-16CaO-24MgO-11Na2O-4Fe2O3) and P40-(40P2O5-16CaO-24MgO-20Na2O), were evaluated for cytotoxicity, cytocompatibility and osteogenic potential for Mesenchymal stem cell (MSC)-based therapy in osteoporotic patients. Evaluations were performed using direct-contact and indirect-contact bone marrow derived human MSC (hMSC)-based experiments, in addition to material characterisations such as morphology, elemental composition and degradation behaviour, which were correlated to the hMSC experiments. Degradation of microspheres (MS) was measured using a novel method where Scanning Electron micrographs was used to assess the number of MS with surface damage (cracks and peeling effect), over 42 days of degradation in culture medium. Results showed that after 42 days, 2%, 46% and 29% of P45 BGMS, P40 BGMS and P40 PGMS, respectively, had cracks or peeling off surfaces. The results for direct-contact hMSC-experiments showed that P45 BGMS supported 1.4 times more hMSCs than P40 BGMS over 31 days of culture period. However, P45 BGMS were not osteoinductive, possibly due to hydrophobic nature of this glass and its slower dissolution rate. On the other hand, in comparison to P45 BGMS, hMSCs seeded on P40 BGMS showed up to 1.7 times higher alkaline phosphatase (ALP) activity on Day 7, up to 1.5 times more collagen and at least 6 times more Ca deposited in extracellular matrix, in addition to osteocalcin on Day 21 of culture, which strongly indicated the osteoinductive nature of P40 BGMS. This effect was also confirmed through indirect-contact experiments where there was higher collagen and Ca production by hMSCs was observed after 25 days of culture in P40 BGMS-conditioned medium as compared to control (no MS) or P45-conditioned medium. Elemental analysis using Energy Dispersive X-Ray Spectroscopic (EDS) analysis revealed that the Ca-based porogen used in the manufacturing of PGMS, may have been retained on the edges of the pores in PGMS. Therefore, an acid-washing step was introduced at the end of manufacturing process in order to remove the porogen and limit the possible cytotoxic effect of porogen and excess calcium. Characterisation results indicated that acid washing changed the physicality of these microspheres without changing their chemical composition. For example, mean and mode pore window sizes on the surface of PGMS increased from 2.63 μm to 2.73 μm and from 1.15 μm to 1.53 μm, respectively, and closed porosity decreased by 27%, as a result of acid washing. However, more detailed EDS analysis revealed that the Ca-based porogen was not being completely removed from PGMS even after acid washing and this may need further investigation. Cytotoxicity evaluations over 7 days of elution (indirect-contact hMSC experiments) suggested that there was marked improvement in hMSC membrane integrity and metabolic activity in PGMS neat extracts after acid washing. Moreover, direct-contact hMSC experiments also showed higher DNA content on acid washed (AW) P40 PGMS over 7 days of culture. Therefore, based on these results, it was hypothesised that acid washing may have opened up some of the pores and removed some of the glass fragments from PGMS surface, which may have been responsible for cytotoxicity in non-AW PGMS. Direct-contact experiments also showed that over 42-day culture period, there was up to 1.6 times higher hMSC numbers in AW P40 PGMS as compared to P40 BGMS. However, this increase was much lower than the expected range as there was more than 10-fold increase in surface area after the introduction of porosity. This was probably due to presence of <5 μm and <10 μm pore window sizes and interconnection sizes, respectively, in these microspheres, which allowed limited penetration of hMSCs into the porous structures. There was also evidence of at least 2 times more ALP activity up to day 42 of culture and up to 1.7 times more collagen production by day 21 of culture, in case of AW P40 PGMS as compared to P40 BGMS, which strongly indicated a positive effect of porosity on osteogenesis. Interestingly, there was also lower Ca and P deposited by hMSCs in porous microspheres, which was in line with the observations made through indirect-contact experiments, where there was lower collagen and Ca production by hMSCs in P40 PGMS-conditioned medium as compared to P40 BGMS-conditioned medium. This negative effect of PGMS was hypothesised due to excess release of glass fragments/particulates and calcium ions into the medium, possibly leading to cytotoxicity. Based on the results shown here, there is a potential of P40 BGMS and AW P40 PGMS for hMSC-based bone repair therapy. However, future work needs to be done in order to limit the delamination of glass surfaces and release of glass fragments/particulates from these MS, as a result of degradation

S-Nitrosoglutathione Acts as a Small Molecule Modulator of Human Fibrin Clot Architecture

<div><h3>Background</h3><p>Altered fibrin clot architecture is increasingly associated with cardiovascular diseases; yet, little is known about how fibrin networks are affected by small molecules that alter fibrinogen structure. Based on previous evidence that S-nitrosoglutathione (GSNO) alters fibrinogen secondary structure and fibrin polymerization kinetics, we hypothesized that GSNO would alter fibrin microstructure.</p> <h3>Methodology/Principal Findings</h3><p>Accordingly, we treated human platelet-poor plasma with GSNO (0.01–3.75 mM) and imaged thrombin induced fibrin networks using multiphoton microscopy. Using custom designed computer software, we analyzed fibrin microstructure for changes in structural features including fiber density, diameter, branch point density, crossing fibers and void area. We report for the first time that GSNO dose-dependently decreased fibrin density until complete network inhibition was achieved. At low dose GSNO, fiber diameter increased 25%, maintaining clot void volume at approximately 70%. However, at high dose GSNO, abnormal irregularly shaped fibrin clusters with high fluorescence intensity cores were detected and clot void volume increased dramatically. Notwithstanding fibrin clusters, the clot remained stable, as fiber branching was insensitive to GSNO and there was no evidence of fiber motion within the network. Moreover, at the highest GSNO dose tested, we observed for the first time, that GSNO induced formation of fibrin agglomerates.</p> <h3>Conclusions/Significance</h3><p>Taken together, low dose GSNO modulated fibrin microstructure generating coarse fibrin networks with thicker fibers; however, higher doses of GSNO induced abnormal fibrin structures and fibrin agglomerates. Since GSNO maintained clot void volume, while altering fiber diameter it suggests that GSNO may modulate the remodeling or inhibition of fibrin networks over an optimal concentration range.</p> </div

GSNO reduces fibrin branchpoint density, but does not alter the fiber density/branchpoint ratio.

<p>Images of human fibrin clots in their native state were acquired using multiphoton microscopy and analyzed using custom designed computer software, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#s2" target="_blank">methods</a>. Panels A,B,C show a multiphoton image with three branch points (B1,B2,B3) and their corresponding contour map and surface plots, respectively. Branch points are readily identifiable in surface plots, where three fibers intersect at the branch junction and the width of one fiber is approximately equal to the sum of the other fibers (F1 = F2+F3). As GSNO concentration increased, branch junctions per area decreased, panel D. However, the ratio of fiber density to branch point density was insensitive to GSNO concentration, panel E. *p<0.05, compared to control. Area is 59×59 um². Colour bars are image intensity. Scale bar is 4.25 um.</p

Fibrin polymerization, GSNO targets and fibrin structural elements imaged by multiphoton microscopy.

<p>Panel A shows a schematic of fiber formation. Fibrinogen is a bisymmetrical molecule consisting of three pairs of polypeptide chains (Aα, Bβ, γ)₂ held together by 29 disulfide bonds. It has a trinodal structure, with central E domain containing N-terminal regions of all six chains connected, by two coiled coils, to two end D domains containing the carboxy-terminal ends of the Bβ and γ chains. The carboxy-terminal portion of the Aα chain folds back from the D domain towards the E domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Ryan1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Cote1" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Weisel1" target="_blank">[49]</a>. Thrombin catalyzes the conversion of fibrinogen to fibrin by cleaving fibrinopeptides A,B from the central E domain. Fibrin monomers self assemble, via complementary E and D domain interactions, forming double stranded half-staggered protofibrils, that branch, elongate, and laterally associate, via released C-terminal α-domains, to form fibrin fibers that constitute the fibrin network <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Cote1" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Weisel1" target="_blank">[49]</a>. S-nitrosglutathione (GSNO), a low molecular weight endogenous s-nitrosothiol present in blood, targets both fibrinogen altering its secondary structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Akhter1" target="_blank">[29]</a> and the exposed thiol group (R-SH) on factor XIIIa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Catani1" target="_blank">[31]</a> which stabilizes fibrin networks by cross-linking fibrin, panel B. GSNO has no effect on thrombin activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Akhter1" target="_blank">[29]</a>. (solid arrows indicate thrombin targets, while dashed lines indicate GSNO targets.) Clotting conditions control fibrin clot architecture <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Ryan1" target="_blank">[8]</a> between extreme forms of a fine (thin) fiber and dense network or a thick fiber and coarse (sparse) network, panel B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Ferry1" target="_blank">[14]</a>. Panel C shows multiphoton images of fibrin network structural elements, including fibrin fibers (i), fiber branch junctions (ii), crossing fibers (iii), fibrin clusters (iv) and fibrin agglomerates (v). Fibrinogen schematic adapted from Undas et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone.0043660-Undas2" target="_blank">[50]</a> Scale bars are 700 nm.</p

Crossing fibers are more sensitive than fiber density to GSNO.

<p>Images of native human fibrin clots were acquired using multiphoton microscopy and analyzed using custom designed computer software, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#s2" target="_blank">methods</a>. Panels A,B,E,F show surface plots of fibrin fibers observed in the corresponding multiphoton image and contour figure, panels C,D respectively. Arrows connect the regions of interest. When two fibers cross at a point in space, there is a >40+/−15% increase in fluorescence intensity at the point of contact, that is proportional to fiber intensity. Panel A shows a trifunctional junction (F1 = F2+F3), panel B shows one fiber (F2) passing beneath another (F1), panel E shows two fibers (F1 and F2) crossing and panel F shows six fiber segments (or possibly three fibers) crossing at a point. As GSNO concentration increased, crossing fibers per area decreased, panel G. The ratio of fiber density to crossing fibers increased with increasing GSNO concentration, indicating the decrease in crossing fibers was greater than the decrease in fiber density, panel H. *p<0.05, compared to control. Area is 59×59 um². Colour bars are image intensity. Scale bars are 700 nm.</p

GSNO increases human fibrin clot void volume.

<p>Human fibrin clots were prepared and imaged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#s2" target="_blank">methods</a>. 2D multiphoton images contain spatial information that can be used to quantify clot void volume and projected clot void area from a stack of three images. The calculation uses binary images of clots acquired at 40,50,60 microns above the clot surface, where white pixels are fibers and black pixels are empty space. Panel A shows 3 image stacks for control (C₁,C₂,C₃) and 2.5 mM GSNO (B₁,B₂,B₃), resulting in projected image C and B, respectively. Both clot void volume and projected clot area were insensitive to GSNO at low concentrations, but increased once GSNO exceeded 1.7 mM, panel D. At 3.75 mM GSNO, only fibrin agglomerates were present in plasma, rendering clot void volume effectively 100%, panel D. *p<0.05, compared to control.Scale bars are 8.5 um.</p

GSNO alters human fibrin fiber density and fiber diameter.

<p>Platelet-poor plasma was incubated with GSNO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#s2" target="_blank">methods</a>. Images of native fibrin clots were acquired using multiphoton microscopy and analyzed using custom designed computer software. Panels A,B,C,G show fibrin clot “read out” images at 0,1,2.5,3.75 mM GSNO, respectively. GSNO decreased fibrin fiber density, panel D, but increased fiber diameter to a maximum at 1 mM GSNO, panel E. Panel F plots fibrin density against fiber diameter. Panel C contained abnormal fibrin clusters with numerous thin diameter fibers protruding from a high intensity core. They decreased the average fiber diameter and shifted the expected density-diameter relationship panel F, point C (2.5 mM GSNO). Fibrin agglomerates were detected at the highest GSNO concentration tested, panel G. Fibrin clot parameters displayed on clot “read out” images: n (number of fibers), fiber density (fibers/100 um), fiber intensity (au, mean and CV), fiber diameter (nm, mean or median and CV), fiber thin-thick ratio vs control (diam-TTR (ctrl)), void area (%), crossing fibers (xfibers/fiber), trifunctional junctions (xbranching/fiber), fibrinogen clusters (FbgClusters/mm²) and fibrin agglomerates (AG/mm²). *p<0.05 vs control. Area is 59×59 um². Colour bars are image intensity. Scale bars are 8.5 um.</p

High concentrations of GSNO induced formation of abnormal fibrin clusters and fibrin agglomerates.

<p>Human fibrin clots were prepared and imaged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#s2" target="_blank">methods</a>. When platelet-poor plasma was incubated with 2.5 mM GSNO, image analysis revealed the presence of abnormal fibrin clusters, characterized by high intensity cores with numerous protruding fibers, panels A and B. An unexpected finding was that 3.75 mM GSNO resulted in the formation of fibrin agglomerates, panel C. Panel D is a histogram of agglomerate long axis diameter. A projected binary image from 30,50,70 microns within the plasma sample, revealed the heterogeneous nature of the agglomerates, panel E. See data supplement <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043660#pone-0043660-g003" target="_blank">figure 3</a> for additional images of agglomerates. Based on the GSNO dose-response curves, panel F, fibrin cluster formation preceded fibrin agglomerate formation. *p<0.05, compared to control. Scale bars for panels A,E are 8.5 um. Scale bars for panels B,C are 4.25 um.</p

Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine–Guanosine Solution

<div><p>Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG) are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1) adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2) adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3) guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with <i>in silico</i> analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives.</p></div

Pathway reactions in the mathematical model of PAGGGM-stored RBCs.

<p>Nodes indicate metabolites or ions, and edges indicate enzymatic reactions or transport processes, which were divided into 3 groups (<i>red, blue, gray boxes</i>) by the difference in sensitivity of enzymatic or reaction activity to temperature or pH as described in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone.0071060-Nishino1" target="_blank">[9]</a>. Abbreviations used in this figure are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071060#pone-0071060-t001" target="_blank">Table 1</a>.</p