Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue

Background: The utility of mesenchymal stromal cells (MSCs) in therapeutic applications for regenerative medicine has gained much attention. Clinical translation of MSC-based approaches requires in vitro culture-expansion to achieve a sufficient number of cells. The ideal cell culture medium should be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. Methods: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three different commercially available hPL fulfilling good manufacturing practice criteria for clinical use. BMSCs and ASCs cultured in Minimum Essential Medium Eagle-alpha supplemented with 5% PLT-Max (Mill Creek), Stemulate? PL-S and Stemulate? PL-SP (COOK General Biotechnology) were compared to standard culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. Results: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS medium. In general, the immunophenotype of both BMSCs and ASCs fulfilled International Society for Cellular Therapy (ISCT) criteria after hPL media expansion. Comparative genomic hybridization measurements demonstrated no unbalanced chromosomal rearrangements for BMSCs or ASCs cultured in hPL media or FBS medium. The BMSCs and ASCs could differentiate into osteogenic, adipogenic, or chondrogenic lineages in all four culture conditions. Conclusion: All three clinically approved commercial human platelet lysates accelerated proliferation of BMSCs and ASCs and the cells meet the ISCT mesenchymal phenotypic requirements without exhibiting chromosomal aberrations.     

Influence of patient related factors on number of mesenchymal stromal cells reached after in vitro culture expansion for clinical treatment

Background: Number of stromal cells injected in patients with ischaemic heart disease (IHD) may be of importance for the treatment efficacy, which in turn may be influenced by various patient-related factors. In this study, we investigate whether patient-related factors influence the number of autologous stromal cells reached after in vitro culture expansion for clinical therapy.

Methods: Culture expansion data from 111 patients with IHD treated with autologous stromal cells in three clinical trials were used. We correlated the final cell count after two passages of cultivation with different patient factors.

Results: There was a significant relation between body mass index (BMI) and the number of adipose derived stromal cells (ASCs) reached after culture expansion and for all patients included into the three studies (r?=?0.375, p?=?.019 and r?=?0.200, p?=?.036, respectively). Moreover, there was a significantly higher number of ASCs reached in patients with hypertension compared to those without hypertension and for all patients overall (68.8?±?39.6?×?10⁶ vs. 39.1?±?23.6?×?10⁶, p?=?.020 and 62.0?±?55.0?×?10⁶ vs. 29.0?±?19.3?×?10⁶, p?<?.001, respectively). The same tendency was seen with bone marrow derived mesenchymal stromal cells (MSCs) in patients with hypertension compared to those without hypertension (58.4?±?61.8?×?10⁶ vs. 22.6?±?13.3?×?10⁶, p?<?.001) and in males compared to females (56.4?±?61.5?×?10⁶ vs. 30.9?±?27.9?×?10⁶, p?=?.041). Moreover, a significant negative correlation between left ventricular ejection fraction and number of MSCs was found (r?=??0.287, p?=?.017).

Conclusions: Patient related factors such as BMI, hypertension and gender may influence the number of MSCs reached after in vitro culture expansion.     

Extracellular matrix enhances differentiation of adipose stem cells from infrapatellar fat pad toward chondrogenesis

The objective was to improve proliferation and chondrogenic potential of adipose stem cells (ASCs) by expansion on extracellular matrix (ECM) deposited by either ASCs or synovium‐derived stem cells (SDSCs). ASCs isolated from porcine infrapatellar fat pad were separately expanded on conventional plastic flasks, ASC‐deposited ECM and SDSC‐deposited ECM. ASCs were centrifuged to form pellets and cultured in a serum‐free chondrogenic medium with either TGFβ3 or TGFβ3 combined with BMP‐6. Cell number yielded on ECM expansion did not show a significant difference in deposition between ASCs and SDSCs but was 6–10 times that grown on non‐coated flasks. ECM‐expanded ASCs exhibited a lower level of intracellular reactive oxygen species (ROS) compared to those grown on non‐coated flasks. Typical chondrogenic markers, including type II collagen and glycosaminoglycans (GAGs), were intensively distributed in the pellets from ECM‐expanded ASCs instead of those from flask‐grown cells. ASCs expanded on ECM, either from ASCs or SDSCs, exhibited a similar chondrogenic index (GAG:DNA), which was significantly higher than that from ASCs grown on non‐coated flasks. The combination of TGFβ3 and BMP‐6 increased 36% more in ASC chondrogenic index than the treatment with TGFβ3 alone. Interestingly, ECM pretreatment also decreased expanded ASC hypertrophic marker genes. ECM deposited by either ASCs or SDSCs did not exhibit enhanced adipogenic differentiation of ASCs. Our study indicates that the sequential application of ECM for cell expansion and combined TGFβ3 with BMP‐6 for chondrogenic differentiation may be a promising approach for ASC‐based cartilage tissue engineering and regeneration. Copyright © 2011 John Wiley & Sons, Ltd.     

Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC‐seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell‐seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL‐supplemented media resulted in rMSC death, by a process attributable to heat‐labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd.     

The kinetics of G-CSF mobilization of CD34+ cells in healthy people

When healthy people are given granulocyte colony stimulating factor (G-CSF) for 10 days the number of CD34+ cells in the peripheral blood begins to increase on the fourth day, reaches a maximum on the sixth day and then decreases. In this study, we further define the time and variability of peak mobilization of CD34+ cells. Twenty-two healthy people were given G-CSF (7.5 or 10 μg kg^?1 day^?1) subcutaneously each morning for 5 days and peripheral blood CD34+ cell counts were analysed immediately prior to the fourth (day 4) and fifth (day 5) G-CSF injection and 24 h after the fifth injection (Day 6). White blood cell (WBC) and neutrophil counts were greatest on day 6 [WBC = 43.8 ± 13.9 × 10⁹ L^?1 (mean ± 1 SD) and neutrophils = 36.6 ± 12.8 × 10⁹ L^?1]. In contrast the CD34+ cell counts on day 6 (107 ± 104 × 10⁶ L^?1) were less than on day 5 (128 ± 136 × 10⁶ L^?1) (P= 0.048) but still greater than on day 4 (60.7 ± 40.2 × 10⁶ L^?1) (P < 0.0001). The CD34+ cell counts of 10 donors were measured 2, 4 and 6 h after the fifth injection to determine if the counts increased further between days 5 and 6. The number of CD34+ cells in the blood on day 5 2 h after the fifth injection (193 ± 277 × 10⁶ L^?1) was greater than the number prior to the injection (158 ± 190 × 10⁶ L^?1), 4 h post-injection (139 ± 158 × 10⁶ L^?1) and 6 h post-injection (170 ± 236 × 10⁶ L^?1), but the differences were not significant (P= 0.29, 0.25 and 0.45). The number of CD34+ cells in the blood of 12 people were measured before and after the fourth G-CSF dose. Prior to the day 4 injection the CD34+ count was 61 ± 40 × 10⁶ L^?1. At 2, 4 and 6 h the counts were 60 ± 40, 61 ± 29 and 64 ± 30 × 10⁶ L^?1, respectively, and the differences were not significant (P= 0.99, P= 0.98, and P= 0.73). In conclusion, when healthy volunteers are given daily G-CSF injections, the number of mobilized CD34+ cells was the greatest on day 5, slightly less on day 6 and the least on day 4. If only one PBSC component is needed, PBSCs can be collected on day 5 after only 4 days of G-CSF. If PBSC components are collected on both days 5 and 6, the fifth dose can be given either before or after the collection of the first PBSC component.     

Adipose stromal cells differentiation toward smooth muscle cell phenotype diminishes their vasculogenic activity due to induction of activin A secretion

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell‐contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC‐lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor β1 (TGFβ₁) or activin A has induced expression of SMC markers in ASCs. Analysis performed after treatment withdrawal revealed that secretome of pre‐differentiated ASCs had a reduced potency to support EC survival and these ASCs had diminished ability to support EC vasculogenesis in vitro. Vascularization of subcutaneous implants carrying a mixture of ECs and ASCs was 50% lower when, instead of control, pre‐differentiated ASCs were used. Pre‐differentiated ASCs had an inferior mitogenic response to EC‐produced factors. Differentiation of ASCs was accompanied by upregulation of vascular endothelial growth factor and a decrease in hepatocyte growth factor (HGF) production; however, addition of HGF to the co‐culture incubation media did not improve vasculogenesis. In parallel, ASC treatment with TGFβ₁ induced secretion of activin A. Augmenting co‐culture incubation media with anti‐activin A IgG restored the ability of pre‐differentiated ASCs to support vasculogenesis to the same degree as control ASCs. The present study suggests that TGFβ₁ or activin A‐induced ASC commitment to SMC phenotype negatively affects the ability of ASCs to support EC vasculogenesis in applications based on EC and ASC co‐injection into target tissues. Copyright © 2016 John Wiley & Sons, Ltd.     

Increased survival of human free fat grafts with varying densities of human adipose‐derived stem cells and platelet‐rich plasma

The high absorption rate of transplanted fat has limited the application of autogenous fat grafts in the clinical setting. Therefore, this study aimed to evaluate the effects of platelet‐rich plasma (PRP) and adipose‐derived stem cells (ASCs) on fat regeneration by investigating the impact of PRP and conditioned medium on the biological characteristics of ASCs. Fat grafts were prepared with ASCs at densities of 10⁷/ml, 10⁶/ml, 10⁵/ml, 10⁴/ml and 0/ml with and without PRP and injected subcutaneously into nude mice. Liquid overflow method, haematoxylin and eosin staining, and immunohistochemical analyses were used to examine the fat grafts. The residual fat volume of the 10⁵/ml ASC + PRP group was significantly higher than that of other treatment conditions after 90 days. Furthermore, histological examination revealed that in 10⁵/ml ASCs‐treated grafts normal adipocyte area and capillary formation were increased dramatically compared with other treatment conditions. It is concluded that fat grafts consisting of PRP and 10⁵/ml ASCs constitute an ideal transplant strategy, which may result in decreased absorption and accelerated fat regeneration. This simple and reliable method could provide a valuable and needed tool in plastic and reconstructive surgery. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of two blood cell separators in collecting peripheral blood stem cell components

To ensure that a sufficient number of CD34+ cells are collected for an allogeneic blood progenitor cell transplant, the most effective blood cell separator should be used to collect peripheral blood stem cell (PBSC) components. We compared the effectiveness of two blood cell separators. We gave 29 healthy people 7.5 or 10 μg kg^?1 of granulocyte colony stimulating factor (G-CSF) daily for 5 days and collected one PBSC component with either a Fenwal CS3000 (n = 15) or a Cobe Spectra (n = 14) blood cell separator. The volume of blood processed was the same for each machine (8.4 ± 1.0 L; range = 4.9–9.4 L for the CS3000 and 8.9 ± 1.0 L; range 6.7–10.9 L; P = 0.71). The components collected with the CS3000 contained more mononuclear cells (39.6 ± 21.9 × 10⁹ compared with 26.9 ± 5.6 × 10⁹, P = 0.02) and fewer neutrophils (1.38 ± 1.88 × 10⁹ compared with 5.53 ± 8.71 × 10⁹, = 0.001). The total number of CD34+ cells collected with the two instruments was the same (470 ± 353 × 10⁶ for the CS3000 and 419 ± 351 × 10⁶ for the Spectra; P = 0.64) as was the number of CD34+ cells collected per litre of whole blood processed (55.9 ± 42.0 × 10⁶ L^?1 compared with 45.9 ± 37.9 × 10⁶ L^?1; P = 0.59). The mononuclear cell collection efficiency was greater for the CS3000 (82.4 ± 54.9% compared with 53.3 ± 14.1; P = 0.04) but the CD34+ cell collection efficiencies were the same (87.4 ± 61.1% for the CS3000 compared with 56.3 ± 23.5% for the Spectra; P = 0.07). In conclusion, both blood cell separators collected components which contained large numbers of CD34+ cells, but those collected with the CS3000 contained fewer neutrophils and the CS3000 was more efficient at collecting mononuclear cells.     

Effects of resveratrol on enrichment of adipose‐derived stem cells and their differentiation to osteoblasts in two‐and three‐dimensional cultures

The goal of this study was to develop a method for increasing the yield of multipotent adipose‐derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271⁺, CD73⁺ and CD45^–) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11⁺ and OCN⁺. Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µ m ) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two‐dimensional (2D) cultures as a function of alkaline phosphatase‐specific activity and osteocalcin production. In addition, cells were seeded onto three‐dimensional (3D) poly‐ε‐caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro‐CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol‐treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Clinical‐scale expansion of adipose‐derived stromal cells starting from stromal vascular fraction in a single‐use bioreactor: proof of concept for autologous applications

Adipose‐derived stromal cells (ASCs) are adult multipotent cells increasingly used for cell therapy due to their differentiation potential, their paracrine effect and their convenience. ASCs are currently selected from stromal vascular fractions (SVFs) of adipose tissue and expanded in 2D flasks following good manufacturing practices. This process is limited in surface area, labour‐intensive and expensive, especially for autologous applications requiring selection and expansion steps for every patient. Closed and automated bioreactors offer an alternative for scalable and cost‐effective production of ASCs. This study investigated a single‐use stirred‐tank bioreactor that can expand ASCs from SVFs on microcarriers. A preliminary microcarrier screening in static and spinner flask conditions was performed to evaluate the best candidate for adhesion, amplification and harvest. The selected microcarrier was used for process development in the bioreactor. The first experiments showed poor selectivity and growth of the ASCs from the SVF (n  =  2). The process was then adjusted by two means: (1) decreasing the platelet lysate in the medium for enhancing cell adherence; and (2) adding a shear protectant (Pluronic F68). Following these modifications, we demonstrated that the number of population doublings of ASCs from SVFs was not significantly different between the bioreactor and the 2D controls (n  =  3). In addition, the ASC characterization after culture showed that cells maintained their clonogenic potential, phenotype, differentiation potential and immunosuppressive capacities. This study provides the proof of concept that isolation and amplification of functional ASCs from SVFs can be performed in a stirred‐tank bioreactor combined with microcarriers. Copyright © 2016 John Wiley & Sons, Ltd.     

Industrial approach in developing an advanced therapy product for bone repair

Mesenchymal stem cells (MSCs) are multipotent cells with therapeutic applications. The aim of our work was to develop an advanced therapy product for bone repair, associating autologous human adipose‐derived MSCs (ASCs) with human bone allograft (TBF; Phoenix^®). We drew up specifications that studied: (a) the influence of tissue collection procedures (elective liposuction or non‐invasive resection) and patient age on cell number and function; (b) monolayer cell culture conditions and osteodifferentiation and particularly the possibility of reducing stages of culture; and (c) the bone construct preparation and especially the comparison between two types of cells seeded on bone allograft (number of cultured processed lipoaspirate (PLA) cells and monolayer‐expanded ASCs) and cultured for 1, 2 and 3 weeks. The results showed that tissue harvesting techniques and patient age did not affect PLA cell number and ASC cloning efficiency. PLA cells can be directly osteodifferentiated (instead of culturing them in expansion medium first and then differentiating them) and these cells were able to mineralize when they were cultured in an osteogenic medium containing calcium chloride. PLA cells directly seeded on bone allograft for a minimum of 3 weeks of culture in this osteogenic medium expressed osteocalcin and colonized the matrix better than monolayer‐expanded ASCs. This work detailed the specifications of a pharmaceutical laboratory to develop an advanced therapy product and this current approach is promising for bone repair. Copyright © 2009 John Wiley & Sons, Ltd.     

Temporal profiling of the growth and multi‐lineage potentiality of adipose tissue‐derived mesenchymal stem cells cell‐sheets

Cell‐sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold‐free constructs. Adipose tissue‐derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow‐derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC‐CSs) is non‐existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC‐CSs development; and (b) the multipotentiality of ASC‐CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid‐supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC‐CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT–PCR and histology at multiple time points. ASC‐CSs remained healthy up to 3 weeks of hyperconfluent culture. One week‐old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non‐hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC‐CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.     

Long‐term in vitro expansion of human adipose‐derived stem cells showed low risk of tumourigenicity

In the field of cell‐based therapy and regenerative medicine, clinical application is the ultimate goal. However, one major concern is: does in vitro manipulation during culture expansion increases tumourigenicity risk on the prepared cells? Therefore, the aim of this study was to investigate the effect of long‐term in vitro expansion on human adipose‐derived stem cells (ASCs). The ASCs were harvested from lipo‐aspirate samples and cultured until passage 20 (P20), using standard culture procedures. ASCs at P5, P10, P15 and P20 were analysed for morphological changes, DNA damage (Comet assay), tumour suppressor gene expression level (quantitative PCR), p53 mutation, telomerase activity, telomere length determination and in vivo tumourigenicity test. Our data showed that ASCs lost their fibroblastic feature in long‐term culture. The population doubling time of ASCs increased with long‐term culture especially at P15 and P20. There was an increase in DNA damage at later passages (P15 and P20). No significant changes were observed in both p53 and p21 genes expression throughout the long‐term culture. There was also no p53 mutation detected and no significant changes were recorded in the relative telomerase activity (RTA) and mean telomere length (TRF) in ASCs at all passages. In vivo implantation of ASCs at P15 and P20 into the nude mice did not result in tumour formation after 4 months. The data showed that ASCs have low risk of tumourigenicity up to P20, with a total population doubling of 42 times. This indicates that adipose tissue should be a safe source of stem cells for cell‐based therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

20-MHz Ultrasound for Measurements of Flow-Mediated Dilation and Shear Rate in the Radial Artery

A high-frequency scanning system consisting of a 20-MHz linear array transducer combined with a 20-MHz pulsed Doppler probe was introduced to evaluate the degree of radial artery flow-mediated dilation (FMD [%]) in two groups of patients after 5?min of controlled forearm ischemia followed by reactive hyperemia. In group I, comprising 27 healthy volunteers, FMD (mean?±?standard deviation) was 15.26?±?4.90% (95% confidence interval [CI]: 13.32%–17.20%); in group II, comprising 17 patients with chronic coronary artery disease, FMD was significantly less at 4.53?±?4.11% (95% CI: 2.42%–6.64%). Specifically, the ratio FMD/SR (mean?±?standard deviation), was equal to 5.36?×?10^?4?±?4.64?×?10^?4 (95% CI: 3.54?×?10^?4 to 7.18?×?10^?4) in group I and 1.38?×?10^?4?±?0.89?×?10^?4 (95% CI: 0.70?×?10^?4 to 2.06?×?10^?4) in group II. Statistically significant differences between the two groups were confirmed by a Wilcoxon–Mann–Whitney test for both FMD and FMD/SR (p?<0.01). Areas under receiver operating characteristic curves for FMD and FMD/SR were greater than 0.9. The results confirm the usefulness of the proposed measurements of radial artery FMD and SR in differentiation of normal patients from those with chronic coronary artery disease.     

Expanded adipose-derived stem cells for the treatment of complex perianal fistula including Crohn's disease

Background: Complex perianal fistulising disease is a distressing condition. In patients without Crohn's disease, surgery is the mainstay treatment but faecal incontinence and recurrence are high. Infliximab is used in Crohn's patients but not all respond to therapy. Objective: After an evaluation of the current treatment options, we discuss studies of adipose-derived stem cell (ASC) therapy, a novel approach for treating complex perianal fistulas. Methods: ASCs are obtained from a liposuction procedure and a subsequent expansion process. They are administered according to a strict protocol which involves infusion of the cells into the target lesion along with fibrin glue. Results/conclusions: A Phase IIb study comparing ASC and fibrin glue therapy with fibrin glue therapy alone showed that ASCs were effective at inducing healing in complex perianal fistulas.     

Effect of transcatheter aortic valve implantation on the ascending aorta’s elasticity

Background

The elastic properties of the ascending aorta were studied before and 1?week after transcatheter aortic valve implantation (TAVI). Previous studies have shown that the distensibility of the ascending aorta was decreased in the early post-operative period after aortic valve replacement. Aortic stiffness is a major moderator of arterio-ventricular coupling and an independent predictor of cardiovascular risk and mortality. We evaluated the effect of TAVI on the elastic properties of the ascending aorta in the early post-operative period.

Methods

Aortic distensibility (AD) and Aortic Stiffness Index (ASI) were evaluated using echocardiographic techniques and brachial artery pressure obtained by sphygmomanometry 2–3?days before and 7–8?days after TAVI.

Results

A total of 30 patients (14 males) were studied with a mean age of 79.9?±?4.7?years and aortic valve area before TAVI of 0.61?±?0.16?cm². Mean arterial pressure decreased significantly after TAVI (from 89.6?±?8.9?mmHg to 83.3?±?10.9?mmHg, p?=?0.004). AD did not change significantly after TAVI (pre: 1.89?±?1.11?cm²/(dynes?×?10⁶), post: 2.05?±?1.50?cm²/(dynes?×?10⁶); p?=?0.813). ASI also remained unchanged (pre: 11.4?±?6.5, post: 15.6?±?14.9; p?=?0.349).

Conclusions

The elastic properties of the ascending aorta did not change significantly in the early post-procedural period after TAVI. This may in part be attributable to the less invasive procedure (compared to aortic valve replacement) which has no effect on vasa vasorum flow.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of Temperature on the Size Distribution,Shell Properties,and Stability of Definity®

Physical characterization of an ultrasound contrast agent (UCA) aids in its safe and effective use in diagnostic and therapeutic applications. The goal of this study was to investigate the impact of temperature on the size distribution, shell properties, and stability of Definity^®, a U.S. Food and Drug Administration-approved UCA used for left ventricular opacification. A Coulter counter was modified to enable particle size measurements at physiologic temperatures. The broadband acoustic attenuation spectrum and size distribution of Definity^® were measured at room temperature (25 °C) and physiologic temperature (37 °C) and were used to estimate the viscoelastic shell properties of the agent at both temperatures. Attenuation and size distribution was measured over time to assess the effect of temperature on the temporal stability of Definity^®. The attenuation coefficient of Definity^® at 37 °C was as much as 5?dB higher than the attenuation coefficient measured at 25 °C. However, the size distributions of Definity^® at 25 °C and 37 °C were similar. The estimated shell stiffness and viscosity decreased from 1.76?±?0.18 N/m and 0.21?×?10^–6?±?0.07?×?10^–6 kg/s at 25 °C to 1.01?±?0.07 N/m and 0.04?×?10^–6?±?0.04?×?10^–6 kg/s at 37 °C, respectively. Size-dependent differences in dissolution rates were observed within the UCA population at both 25 °C and 37 °C. Additionally, cooling the diluted UCA suspension from 37 °C to 25 °C accelerated the dissolution rate. These results indicate that although temperature affects the shell properties of Definity^® and can influence the stability of Definity^®, the size distribution of this agent is not affected by a temperature increase from 25 °C to 37 °C.     

Absolute immature platelet counts in the setting of suspected heparin-induced thrombocytopenia may predict anti-PF4-heparin immunoassay testing results

Background

Heparin-induced-thrombocytopenia (HIT) is a disease mediated by antibodies to platelet factor 4 (PF4)-heparin complexes. Immature platelet fraction (%-IPF) and absolute immature platelet count (A-IPC) measure newly-released platelets into circulation and can prove useful in differentiating patients with thrombocytopenic presentations due to consumptive or hypoproduction processes. Therefore, we evaluated utility of A-IPC in a cohort of thrombocytopenic patients suspected of HIT.