Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.

Neural network analysis identifies scaffold properties necessary for in vitro chondrogenesis in elastin-like polypeptide biopolymer scaffolds.

The successful design of biomaterial scaffolds for articular cartilage tissue engineering requires an understanding of the impact of combinations of material formulation parameters on diverse and competing functional outcomes of biomaterial performance. This study sought to explore the use of a type of unsupervised artificial network, a self-organizing map, to identify relationships between scaffold formulation parameters (crosslink density, molecular weight, and concentration) and 11 such outcomes (including mechanical properties, matrix accumulation, metabolite usage and production, and histological appearance) for scaffolds formed from crosslinked elastin-like polypeptide (ELP) hydrogels. The artificial neural network recognized patterns in functional outcomes and provided a set of relationships between ELP formulation parameters and measured outcomes. Mapping resulted in the best mean separation amongst neurons for mechanical properties and pointed to crosslink density as the strongest predictor of most outcomes, followed by ELP concentration. The map also grouped formulations together that simultaneously resulted in the highest values for matrix production, greatest changes in metabolite consumption or production, and highest histological scores, indicating that the network was able to recognize patterns amongst diverse measurement outcomes. These results demonstrated the utility of artificial neural network tools for recognizing relationships in systems with competing parameters, toward the goal of optimizing and accelerating the design of biomaterial scaffolds for articular cartilage tissue engineering.

Trade-offs between cancer and other diseases: do they exist and influence longevity?

Relationships between aging, disease risks, and longevity are not yet well understood. For example, joint increases in cancer risk and total survival observed in many human populations and some experimental aging studies may be linked to a trade-off between cancer and aging as well as to the trade-off(s) between cancer and other diseases, and their relative impact is not clear. While the former trade-off (between cancer and aging) received broad attention in aging research, the latter one lacks respective studies, although its understanding is important for developing optimal strategies of increasing both longevity and healthy life span. In this paper, we explore the possibility of trade-offs between risks of cancer and selected major disorders. First, we review current literature suggesting that the trade-offs between cancer and other diseases may exist and be linked to the differential intensity of apoptosis. Then we select relevant disorders for the analysis (acute coronary heart disease [ACHD], stroke, asthma, and Alzheimer disease [AD]) and calculate the risk of cancer among individuals with each of these disorders, and vice versa, using the Framingham Study (5209 individuals) and the National Long Term Care Survey (NLTCS) (38,214 individuals) data. We found a reduction in cancer risk among old (80+) men with stroke and in risk of ACHD among men (50+) with cancer in the Framingham Study. We also found an increase in ACHD and stroke among individuals with cancer, and a reduction in cancer risk among women with AD in the NLTCS. The manifestation of trade-offs between risks of cancer and other diseases thus depended on sex, age, and study population. We discuss factors modulating the potential trade-offs between major disorders in populations, e.g., disease treatments. Further study is needed to clarify possible impact of such trade-offs on longevity.

Ligament-derived matrix stimulates a ligamentous phenotype in human adipose-derived stem cells.

Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p

Effect of microRNA modulation on bioartificial muscle function.

Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle tissues. As assessed by functional force measurements in response to electrical stimulation at frequencies ranging from 0 to 20 Hz, peak forces exhibited by BAMs with miR-133 inhibition (anti-miR-133) were on average 20% higher than the corresponding negative control, although dynamic responses to electrical stimulation in miRNA-transfected BAMs and negative controls were similar to nontransfected controls. Immunostaining for alpha-actinin and myosin also showed more distinct striations and myofiber organization in anti-miR-133 BAMs, and fiber diameters were significantly larger in these BAMs over both the nontransfected and negative controls. Compared to the negative control, anti-miR-133 BAMs exhibited more intense nuclear staining for Mef2, a key myogenic differentiation marker. To our knowledge, this study is the first to demonstrate that miRNA mediation has functional effects on tissue-engineered constructs.

Functional properties of cell-seeded three-dimensionally woven poly(epsilon-caprolactone) scaffolds for cartilage tissue engineering.

Articular cartilage possesses complex mechanical properties that provide healthy joints the ability to bear repeated loads and maintain smooth articulating surfaces over an entire lifetime. In this study, we utilized a fiber-reinforced composite scaffold designed to mimic the anisotropic, nonlinear, and viscoelastic biomechanical characteristics of native cartilage as the basis for developing functional tissue-engineered constructs. Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were encapsulated with a fibrin hydrogel, seeded with human adipose-derived stem cells, and cultured for 28 days in chondrogenic culture conditions. Biomechanical testing showed that PCL-based constructs exhibited baseline compressive and shear properties similar to those of native cartilage and maintained these properties throughout the culture period, while supporting the synthesis of a collagen-rich extracellular matrix. Further, constructs displayed an equilibrium coefficient of friction similar to that of native articular cartilage (mu(eq) approximately 0.1-0.3) over the prescribed culture period. Our findings show that three-dimensionally woven PCL-fibrin composite scaffolds can be produced with cartilage-like mechanical properties, and that these engineered properties can be maintained in culture while seeded stem cells regenerate a new, functional tissue construct.

Evaluating intra-articular drug delivery for the treatment of osteoarthritis in a rat model.

Osteoarthritis (OA) is a degenerative joint disease that can result in joint pain, loss of joint function, and deleterious effects on activity levels and lifestyle habits. Current therapies for OA are largely aimed at symptomatic relief and may have limited effects on the underlying cascade of joint degradation. Local drug delivery strategies may provide for the development of more successful OA treatment outcomes that have potential to reduce local joint inflammation, reduce joint destruction, offer pain relief, and restore patient activity levels and joint function. As increasing interest turns toward intra-articular drug delivery routes, parallel interest has emerged in evaluating drug biodistribution, safety, and efficacy in preclinical models. Rodent models provide major advantages for the development of drug delivery strategies, chiefly because of lower cost, successful replication of human OA-like characteristics, rapid disease development, and small joint volumes that enable use of lower total drug amounts during protocol development. These models, however, also offer the potential to investigate the therapeutic effects of local drug therapy on animal behavior, including pain sensitivity thresholds and locomotion characteristics. Herein, we describe a translational paradigm for the evaluation of an intra-articular drug delivery strategy in a rat OA model. This model, a rat interleukin-1beta overexpression model, offers the ability to evaluate anti-interleukin-1 therapeutics for drug biodistribution, activity, and safety as well as the therapeutic relief of disease symptoms. Once the action against interleukin-1 is confirmed in vivo, the newly developed anti-inflammatory drug can be evaluated for evidence of disease-modifying effects in more complex preclinical models.

Chondrogenesis and mineralization during in vitro culture of human mesenchymal stem cells on three-dimensional woven scaffolds.

Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(ɛ-caprolactone) (PCL) scaffolds are promising tools for skeletal tissue engineering. We hypothesized that in vitro culture duration and medium additives can individually and interactively influence the structure, composition, mechanical, and molecular properties of engineered tissues based on hMSCs and 3D poly(ɛ-caprolactone). Bone marrow hMSCs were suspended in collagen gel, seeded on scaffolds, and cultured for 1, 21, or 45 days under chondrogenic and/or osteogenic conditions. Structure, composition, biomechanics, and gene expression were analyzed. In chondrogenic medium, cartilaginous tissue formed by day 21, and hypertrophic mineralization was observed in the newly formed extracellular matrix at the interface with underlying scaffold by day 45. Glycosaminoglycan, hydroxyproline, and calcium contents, and alkaline phosphatase activity depended on culture duration and medium additives, with significant interactive effects (all p < 0.0001). The 45-day constructs exhibited mechanical properties on the order of magnitude of native articular cartilage (aggregate, Young's, and shear moduli of 0.15, 0.12, and 0.033 MPa, respectively). Gene expression was characteristic of chondrogenesis and endochondral bone formation, with sequential regulation of Sox-9, collagen type II, aggrecan, core binding factor alpha 1 (Cbfα1)/Runx2, bone sialoprotein, bone morphogenetic protein-2, and osteocalcin. In contrast, osteogenic medium produced limited osteogenesis. Long-term culture of hMSC on 3D scaffolds resulted in chondrogenesis and regional mineralization at the interface between soft, newly formed engineered cartilage, and stiffer underlying scaffold. These findings merit consideration when developing grafts for osteochondral defect repair.

Optimized, unequal pulse spacing in multiple echo sequences improves refocusing in magnetic resonance.

A recent quantum computing paper (G. S. Uhrig, Phys. Rev. Lett. 98, 100504 (2007)) analytically derived optimal pulse spacings for a multiple spin echo sequence designed to remove decoherence in a two-level system coupled to a bath. The spacings in what has been called a "Uhrig dynamic decoupling (UDD) sequence" differ dramatically from the conventional, equal pulse spacing of a Carr-Purcell-Meiboom-Gill (CPMG) multiple spin echo sequence. The UDD sequence was derived for a model that is unrelated to magnetic resonance, but was recently shown theoretically to be more general. Here we show that the UDD sequence has theoretical advantages for magnetic resonance imaging of structured materials such as tissue, where diffusion in compartmentalized and microstructured environments leads to fluctuating fields on a range of different time scales. We also show experimentally, both in excised tissue and in a live mouse tumor model, that optimal UDD sequences produce different T(2)-weighted contrast than do CPMG sequences with the same number of pulses and total delay, with substantial enhancements in most regions. This permits improved characterization of low-frequency spectral density functions in a wide range of applications.

Do diabetic veterans use the Internet? Self-reported usage, skills, and interest in using My HealtheVet Web portal.

OBJECTIVE: The Veterans Health Administration has developed My HealtheVet (MHV), a Web-based portal that links veterans to their care in the veteran affairs (VA) system. The objective of this study was to measure diabetic veterans' access to and use of the Internet, and their interest in using MHV to help manage their diabetes. MATERIALS AND METHODS: Cross-sectional mailed survey of 201 patients with type 2 diabetes and hemoglobin A(1c) > 8.0% receiving primary care at any of five primary care clinic sites affiliated with a VA tertiary care facility. Main measures included Internet usage, access, and attitudes; computer skills; interest in using the Internet; awareness of and attitudes toward MHV; demographics; and socioeconomic status. RESULTS: A majority of respondents reported having access to the Internet at home. Nearly half of all respondents had searched online for information about diabetes, including some who did not have home Internet access. More than a third obtained "some" or "a lot" of their health-related information online. Forty-one percent reported being "very interested" in using MHV to help track their home blood glucose readings, a third of whom did not have home Internet access. Factors associated with being "very interested" were as follows: having access to the Internet at home (p < 0.001), "a lot/some" trust in the Internet as a source of health information (p = 0.002), lower age (p = 0.03), and some college (p = 0.04). Neither race (p = 0.44) nor income (p = 0.25) was significantly associated with interest in MHV. CONCLUSIONS: This study found that a diverse sample of older VA patients with sub-optimally controlled diabetes had a level of familiarity with and access to the Internet comparable to an age-matched national sample. In addition, there was a high degree of interest in using the Internet to help manage their diabetes.

Conductive junctions with parallel graphene sheets.

The establishment of conductive graphene-molecule-graphene junction is investigated through first-principles electronic structure calculations and quantum transport calculations. The junction consists of a conjugated molecule connecting two parallel graphene sheets. The effects of molecular electronic states, structure relaxation, and molecule-graphene contact on the conductance of the junction are explored. A conductance as large as 0.38 conductance quantum is found achievable with an appropriately oriented dithiophene bridge. This work elucidates the designing principles of promising nanoelectronic devices based on conductive graphene-molecule-graphene junctions.

Toward the development of national telehealth services: the role of Veterans Health Administration and future directions for research.

The Veterans Health Administration (VHA) in the Department of Veteran Affairs (VA) has emerged as a national and international leader in the delivery and research of telehealth-based treatment. Several unique characteristics of care in VA settings intersect to create an ideal environment for telehealth modalities and research. However, the value of telehealth experience and initiatives in VA settings is limited if telehealth strategies cannot be widely exported to other public or private systems. Whereas a hierarchical organization, such as VA, can innovate and fund change relatively quickly based on provider and patient preferences and a growing knowledge base, other health provider organizations and third-party payers may likely require replicable scientific findings over time before incremental investments will be made to create infrastructure, reform regulatory barriers, and amend laws to accommodate expansion of telehealth modalities. Accordingly, large-scale scientifically rigorous telehealth research in VHA settings is essential not only to investigate the efficacy of existing and future telehealth practices in VHA, but also to hasten the development of telehealth infrastructure in private and other public health settings. We propose an expanded partnership between the VA, NIH, and other funding agencies to investigate creative and pragmatic uses of telehealth technology. To this end, we identify six specific areas of research we believe to be particularly relevant to the efficient development of telehealth modalities in civilian and military contexts outside VHA.

Accelerating self-consistent field convergence with the augmented Roothaan-Hall energy function.

Based on Pulay's direct inversion iterative subspace (DIIS) approach, we present a method to accelerate self-consistent field (SCF) convergence. In this method, the quadratic augmented Roothaan-Hall (ARH) energy function, proposed recently by Høst and co-workers [J. Chem. Phys. 129, 124106 (2008)], is used as the object of minimization for obtaining the linear coefficients of Fock matrices within DIIS. This differs from the traditional DIIS of Pulay, which uses an object function derived from the commutator of the density and Fock matrices. Our results show that the present algorithm, abbreviated ADIIS, is more robust and efficient than the energy-DIIS (EDIIS) approach. In particular, several examples demonstrate that the combination of ADIIS and DIIS ("ADIIS+DIIS") is highly reliable and efficient in accelerating SCF convergence.

The effect of hydrogen bonding on the diffusion of water in n-alkanes and n-alcohols measured with a novel single microdroplet method.

While the Stokes-Einstein (SE) equation predicts that the diffusion coefficient of a solute will be inversely proportional to the viscosity of the solvent, this relation is commonly known to fail for solutes, which are the same size or smaller than the solvent. Multiple researchers have reported that for small solutes, the diffusion coefficient is inversely proportional to the viscosity to a fractional power, and that solutes actually diffuse faster than SE predicts. For other solvent systems, attractive solute-solvent interactions, such as hydrogen bonding, are known to retard the diffusion of a solute. Some researchers have interpreted the slower diffusion due to hydrogen bonding as resulting from the effective diffusion of a larger complex of a solute and solvent molecules. We have developed and used a novel micropipette technique, which can form and hold a single microdroplet of water while it dissolves in a diffusion controlled environment into the solvent. This method has been used to examine the diffusion of water in both n-alkanes and n-alcohols. It was found that the polar solute water, diffusing in a solvent with which it cannot hydrogen bond, closely resembles small nonpolar solutes such as xenon and krypton diffusing in n-alkanes, with diffusion coefficients ranging from 12.5x10(-5) cm(2)/s for water in n-pentane to 1.15x10(-5) cm(2)/s for water in hexadecane. Diffusion coefficients were found to be inversely proportional to viscosity to a fractional power, and diffusion coefficients were faster than SE predicts. For water diffusing in a solvent (n-alcohols) with which it can hydrogen bond, diffusion coefficient values ranged from 1.75x10(-5) cm(2)/s in n-methanol to 0.364x10(-5) cm(2)/s in n-octanol, and diffusion was slower than an alkane of corresponding viscosity. We find no evidence for solute-solvent complex diffusion. Rather, it is possible that the small solute water may be retarded by relatively longer residence times (compared to non-H-bonding solvents) as it moves through the liquid.

Structural manifestation of the delocalization error of density functional approximations: C(4N+2) rings and C(20) bowl, cage, and ring isomers.

The ground state structure of C(4N+2) rings is believed to exhibit a geometric transition from angle alternation (N < or = 2) to bond alternation (N > 2). All previous density functional theory (DFT) studies on these molecules have failed to reproduce this behavior by predicting either that the transition occurs at too large a ring size, or that the transition leads to a higher symmetry cumulene. Employing the recently proposed perspective of delocalization error within DFT we rationalize this failure of common density functional approximations (DFAs) and present calculations with the rCAM-B3LYP exchange-correlation functional that show an angle-to-bond-alternation transition between C(10) and C(14). The behavior exemplified here manifests itself more generally as the well known tendency of DFAs to bias toward delocalized electron distributions as favored by Huckel aromaticity, of which the C(4N+2) rings provide a quintessential example. Additional examples are the relative energies of the C(20) bowl, cage, and ring isomers; we show that the results from functionals with minimal delocalization error are in good agreement with CCSD(T) results, in contrast to other commonly used DFAs. An unbiased DFT treatment of electron delocalization is a key for reliable prediction of relative stability and hence the structures of complex molecules where many structure stabilization mechanisms exist.