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Computer Methods in Biomechanics and Biomedical Engineering 11/2012; · 0.85 Impact Factor
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ABSTRACT: A major drawback of mechanical and prosthetic heart valves is their inability to permit somatic growth. By contrast, tissue-engineered pulmonary valves potentially have the capacity to remodel and integrate with the patient. For this purpose, adult stem cells may be suitable. Previously, human periodontal ligament cells (PDLs) have been explored as a reliable and robust progenitor cell source for cardiac muscle regeneration (Pelaez, D. Electronic Thesis and Dissertation Database, Coral Gables, FL, May 2011). Here, we investigate the potential of PDLs to support the valve lineage, specifically the concomitant differentiation to both endothelial cell (EC) and smooth muscle cell (SMC) types. We were able to successfully promote PDL differentiation to both SMC and EC phenotypes through a combination of stimulatory approaches using biochemical and mechanical flow conditioning (steady shear stress of 1 dyne/cm(2)), with flow-based mechanical conditioning having a predominant effect on PDL differentiation, particularly to ECs; in addition, strong expression of the marker FZD2 and an absence of the marker MLC1F point toward a unique manifestation of smooth muscle by PDLs after undergoing steady-flow mechanical conditioning alone, possible by only the heart valve and pericardium phenotypes. It was also determined that steady flow (which was performed using a physiologically relevant [for heart valves] magnitude of ∼5-6 dynes/cm(2)) augmented the synthesis of the extracellular matrix collagen proteins. We conclude that under steady-flow dynamic culture environments, human PDLs can differentiate to heterogeneous cell populations that are relevant to heart valve tissue engineering. Further exploration of human PDLs for this purpose is thus warranted.
Tissue Engineering Part A 09/2012; · 4.64 Impact Factor
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ABSTRACT: Oscillatory shear stress (OSS), caused by time-varying flow environments, may play a critical role in the production of engineered tissue by bone marrow-derived stem cells. This is particularly relevant in heart valve tissue engineering (HVTE), owing to the intense haemodynamic environments that surround native valves. In this study, we examined and quantified the role that (i) physiologically relevant scales of pulsatility and (ii) changes in geometry as a function of specimen flexure have in creating OSS conditions. A U-shaped bioreactor capable of producing flow, stretch and flexure was modelled with housed specimens, and computational fluid dynamic simulations were performed. We found that physiologically relevant OSS can be maximised by the application of pulsatile flow to straight, non-moving specimens in a uniform manner. This finding reduces a substantial layer of complexity in dynamic HVTE protocols in which traditionally, time-varying flow has been promoted through specimen movement in custom-made bioreactors.
Computer Methods in Biomechanics and Biomedical Engineering 08/2012; · 0.85 Impact Factor
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ABSTRACT: The intimal and medial linings of the pulmonary artery consist largely of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs), respectively. The migration of these cell types to a potential tissue-engineered pulmonary valve (TEPV) implant process is therefore of interest in understanding the valve remodeling process. Visualization and cell tracking by MRI, which employs hypointense contrast achievable through the use of superparamagnetic iron oxide (SPIO) microparticles to label cells, provides a method in which this can be studied. We investigated the SPIO labeling efficiency of human VECs and VSMCs, and used two- and three-dimensional gradient echo sequences to track the migration of these cells in agar gel constructs. Protamine sulfate (4.5 µg/mL) was used to enhance SPIO uptake and was found to have no influence on cell viability or proliferation. MRI experiments were initially performed using a 9.4-T scanner. The results demonstrated that the spatial positions of hypointense spots were relatively unchanged over 12 days. Subsequent MR experiments performed at 7 T demonstrated that three-dimensional imaging provided the best spatial resolution to assess cell fate. R(2)* maps were bright in SPIO cell-encapsulated gels in comparison with unlabeled counterparts. Signal voids were ruled out as hypointense regions owing to the smooth exponential decay of T(2)* in these voxels. As a next step, we intend to use the SPIO cell labeling and MR protocols established in this study to assess whether hemodynamic stresses will alter the vascular cell migratory patterns. These studies will shed light on the mechanisms of vascular remodeling after TEPV implantation.
NMR in Biomedicine 03/2012; 25(3):410-7. · 3.21 Impact Factor
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ABSTRACT: Labeling of cells with superparamagnetic iron oxide nanoparticles permits cell tracking by (1)H MRI while (31)P MRS allows non-invasive evaluation of cellular bioenergetics. We evaluated the compatibility of these two techniques by obtaining (31)P NMR spectra of iron-labeled and unlabeled immobilized C2C12 myoblast cells in vitro. Broadened but usable (31)P spectra were obtained and peak area ratios of resonances corresponding to intracellular metabolites showed no significant differences between labeled and unlabeled cell populations. We conclude that (31)P NMR spectra can be obtained from cells labeled with sufficient iron to permit visualization by (1)H imaging protocols and that these spectra have sufficient quality to be used to assess metabolic status. This result introduces the possibility of using localized (31)P MRS to evaluate the viability of iron-labeled therapeutic cells as well as surrounding host tissue in vivo.
NMR in Biomedicine 12/2010; 23(10):1166-72. · 3.21 Impact Factor
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ABSTRACT: Osteoarthritis (OA) is a prevalent age-associated disease involving altered chondrocyte homeostasis and cartilage degeneration. The avascular nature of cartilage and the altered chondrocyte phenotype characteristic of OA severely limit the capacity for in vivo tissue regeneration. Cell- and tissue-based repair has the potential to revolutionize treatment of OA, but those approaches have exhibited limited clinical success to date. In this study, we test the hypothesis that bovine and human chondrocytes in a collagen type I scaffold will form hyaline cartilage ex vivo with immunohistochemical, biochemical, and magnetic resonance (MR) endpoints similar to the original native cartilage. Chondrocytes were isolated from 1- to 3-week-old calf knee cartilage or from cartilage obtained from human total knee arthroplasties, suspended in 2.7 mg/mL collagen I, and plated as 300 microL spot cultures with 5 x 10(6) each. Medium formulations were varied, including the amount of serum, the presence or absence of ascorbate, and treatments with cytokines. Bovine chondrocytes generated metachromatic territorial and interstitial matrix and accumulated type II collagen over time. Type VI collagen was confined primarily to the pericellular region. The ex vivo-formed bovine cartilage contained more chondroitin sulfate per dry weight than native cartilage. Human chondrocytes remained viable and generated metachromatic territorial matrix, but were unable to support interstitial matrix accumulation. MR analysis of ex vivo-formed bovine cartilage revealed evidence of progressively maturing matrix, but MR-derived indices of tissue quality did not reach those of native cartilage. We conclude that the collagen-spot culture model supports formation and maturation of three-dimensional hyaline cartilage from active bovine chondrocytes. Future studies will focus on determining the capacity of human chondrocytes to show comparable tissue formation.
Tissue Engineering Part A 02/2010; 16(7):2183-96. · 4.64 Impact Factor
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ABSTRACT: We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083-95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039-49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (approximately 185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted in substantially larger collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (approximately 7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment.
Biomaterials 11/2009; 31(6):1114-25. · 7.40 Impact Factor
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ABSTRACT: The distribution of cells within tissue-engineered constructs is difficult to study through nondestructive means, such as would be required after implantation. However, cell labeling with iron-containing particles may prove to be a useful approach to this problem, because regions containing such labeled cells have been shown to be readily detectable using magnetic resonance imaging (MRI). In this study, we used the Food and Drug Administration-approved superparamagnetic iron oxide (SPIO) contrast agent Feridex in combination with transfection agents to label chondrocytes and visualize them with MRI in two different tissue-engineered cartilage constructs. Correspondence between labeled cell spatial location as determined using MRI and histology was established. The SPIO-labeling process was found not to affect the phenotype or viability of the chondrocytes or the production of major cartilage matrix constituents. We believe that this method of visualizing and tracking chondrocytes may be useful in the further development of tissue engineered cartilage therapeutics.
Tissue Engineering Part A 09/2009; 15(12):3899-910. · 4.64 Impact Factor
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ABSTRACT: The purpose of this study was to determine if the noninvasive and nondestructive technique of magnetic resonance imaging could be used to quantify the amount of repair tissue that fills surgically-induced chondral defects in the rabbit. Sixteen 4-mm diameter full-thickness chondral defects were created. A photopolymerizable hydrogel was used to seal the defects as a treatment modality. At 5 weeks, the animals were sacrificed and the distal femur was subjected to MRI analyses at high field (9.4 T). The transverse relaxation time (T(2)) in each defect was measured. Histology and histomorphometric analysis were used to quantify the amount of repair tissue that filled each defect. The relationship between T(2) and percent tissue fill was found to fit well to a negatively sloped, linear model. The linear (Pearson's product-moment) correlation coefficient was found to be r = -0.82 and the associated coefficient of determination was r(2) = 0.67. This correlation suggests that the MRI parameter T(2) can be used to track changes in the amount of repair tissue that fills cartilage defects. This would be especially useful in in vivo cartilage tissue engineering studies that attempt to determine optimal biomaterials for scaffold design.
Journal of Biomedical Materials Research Part B Applied Biomaterials 09/2008; 86(2):375-80. · 2.15 Impact Factor
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ABSTRACT: The glycosaminoglycan (GAG) content of engineered cartilage is a determinant of biochemical and mechanical quality. The ability to measure the degree to which GAG content is maintained or increases in an implant is therefore of importance in cartilage repair procedures. The gadolinium exclusion magnetic resonance imaging (MRI) method for estimating matrix fixed charge density (FCD) is ideally suited to this. One promising approach to cartilage repair is use of seeded injectable hydrogels. Accordingly, we assess the reliability of measuring GAG content in such a system ex vivo using MRI. Samples of the photopolymerizable hydrogel, poly(ethylene oxide) diacrylate, were seeded with bovine chondrocytes (approximately 2.4 million cells/sample). The FCD of the constructs was determined using MRI after 9, 16, 29, 36, 43, and 50 days of incubation. Values were correlated with the results of biochemical determination of GAG from the same samples. FCD and GAG were found to be statistically significantly correlated (R2 = 0.91, p < 0.01). We conclude that MRI-derived FCD measurements of FCD in injectable hydrogels reflect tissue GAG content and that this methodology therefore has potential for in vivo monitoring of such constructs.
Tissue Engineering Part C Methods 07/2008; 14(3):243-9. · 4.64 Impact Factor
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ABSTRACT: Assessment of subtle changes in proteoglycan (PG) and collagen, the primary macromolecular components of cartilage, which is critical for diagnosis of the early stages of osteoarthritis (OA), has so far remained a challenge. In this study we induced osteoarthritic cartilage changes in a rabbit model by ligament transection and medial meniscectomy and monitored disease progression by infrared fiber optic probe (IFOP) spectroscopy, Fourier transform infrared imaging spectroscopy (FT-IRIS), and magnetic resonance imaging (MRI) microscopy. IFOP studies combined with chemometric partial least-squares analysis enabled us to monitor progressive cartilage surface changes from two to twelve weeks post-surgery. FT-IRIS studies of histological sections of femoral condyle cartilage revealed that compared with control cartilage the OA cartilage had significantly reduced PG content 2 and 4 weeks post-surgery, collagen fibril orientation changes 2 and 4 weeks post-surgery, and changes in collagen integrity 2 and 10 weeks post-surgery, but no significant changes in collagen content at any time. MR microscopy studies revealed reduced fixed charge density (FCD), indicative of reduced PG content, in the OA cartilage, compared with controls, 4 weeks post-surgery. A non-significant trend toward higher apparent MT exchange rate, k(m), was also found in the OA cartilage at this time point, suggesting changes in collagen structural features. These two MR findings for FCD and k(m) parallel the FT-IRIS findings of reduced PG content and altered collagen integrity, respectively. MR microscopy studies of the cartilage at the 12-week time point also found a trend toward longer T (2) values and reduced anisotropy in the deep zone of the OA cartilage, consistent with increased hydration and less ordered collagen. These studies reveal that FT-IRIS and MR microscopy provide complementary data on compositional changes in articular cartilage in the early stages of osteoarthritic degradation.
Analytical and Bioanalytical Chemistry 04/2007; 387(5):1601-12. · 3.78 Impact Factor
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Joseph A. Baur,
Kevin J. Pearson,
Nathan L. Price,
Hamish A. Jamieson,
Carles Lerin,
Avash Kalra,
Vinayakumar V. Prabhu,
Joanne S. Allard,
Guillermo Lopez-Lluch,
Kaitlyn Lewis, [......],
Kenneth W. Fishbein,
Richard G. Spencer,
Edward G. Lakatta,
David Le Couteur,
Reuben J. Shaw,
Placido Navas,
Pere Puigserver,
Donald K. Ingram,
Rafael de Cabo,
David A. Sinclair
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ABSTRACT: Resveratrol (3,5,4'-trihydroxystilbene) extends the lifespan of diverse species including Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In these organisms, lifespan extension is dependent on Sir2, a conserved deacetylase proposed to underlie the beneficial effects of caloric restriction. Here we show that resveratrol shifts the physiology of middle-aged mice on a high-calorie diet towards that of mice on a standard diet and significantly increases their survival. Resveratrol produces changes associated with longer lifespan, including increased insulin sensitivity, reduced insulin-like growth factor-1 (IGF-I) levels, increased AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor- coactivator 1 (PGC-1) activity, increased mitochondrial number, and improved motor function. Parametric analysis of gene set enrichment revealed that resveratrol opposed the effects of the high-calorie diet in 144 out of 153 significantly altered pathways. These data show that improving general health in mammals using small molecules is an attainable goal, and point to new approaches for treating obesity-related disorders and diseases of ageing.
Nature 10/2006; 444(7117):337-342. · 36.28 Impact Factor
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ABSTRACT: A hydrogel is a highly hydrated polymer gel suitable for use as a scaffold for tissue engineering. One important application is to the repair of cartilage defects due to injury or osteoarthritis. Integration of the hydrogel with surrounding tissue is critical for the long-term functionality of the implant; however direct visualization of integration is difficult and invasive. Accordingly, we used MRI to noninvasively investigate the integration of hydrogel in cartilage. Two integration methods were assessed: (1) cartilage-initiated and (2) chemical, using chondroitin sulphate-methacrylate-aldehyde (CS-MA-ald) as an adhesive. These were compared to a control group, that is, standard, nonintegrated hydrogel photopolymerization. Spatial variation of the transverse relaxation time, T(2), across the transition region was used to determine the effectiveness of integration. In the CS-MA-ald group only, two interfaces were found. This provides evidence of an intermediate adhesive layer between hydrogel and cartilage. Second, the thickness of the transition region between hydrogel to cartilage in the CS-MA-ald group was 1.32 mm as compared to 1.20 mm and 1.17 mm in the tissue-initiated and nonintegrated groups, respectively. We interpret this as a more gradual transition region of hydrogel to cartilage and hence a greater degree of integration when an adhesive layer is present.
Journal of Biomedical Materials Research Part B Applied Biomaterials 05/2006; 77(1):144-8. · 2.15 Impact Factor
