Monolayer cell expansion conditions affect the chondrogenic potential of adipose-derived stem cells

Adipose-derived stem cells (ASCs) are an abundant, readily available population of multipotent progenitor cells that reside in adipose tissue. Isolated ASCs are typically expanded in monolayer on standard tissue culture plastic with a basal medium containing 10% fetal bovine serum. However, recent data suggest that altering the monolayer expansion conditions by using suspension culture plastic, adding growth factors to the medium, or adjusting the seeding density may affect the self-renewal rate, multipotency, and lineage-specific differentiation potential of the ASCs. We hypothesized that variation in any of these expansion conditions would influence the chondrogenic potential of ASCs. ASCs were isolated from human liposuction waste tissue and expanded through two passages with different tissue culture plastic, feed medium, and cell seeding densities. Once expanded, the cells were cast in an agarose gel and subjected to identical chondrogenic culture conditions for 7 days, at which point cell viability, radiolabel incorporation, and gene expression were measured. High rates of matrix synthesis upon chondrogenic induction were mostly associated with smaller cells, as indicated by cell width and area on tissue culture plastic, and it appears that expansion in a growth factor supplemented medium is important in maintaining this morphology. All end-point measures were highly dependent on the specific monolayer culture conditions. These results support the hypothesis that monolayer culture conditions may "prime" the cells or predispose them towards a specific phenotype and thus underscore the importance of early culture conditions in determining the growth and differentiation potential of ASCs.     

Site-specific effects of compression on macromolecular diffusion in articular cartilage

Articular cartilage is the connective tissue that lines joints and provides a smooth surface for joint motion. Because cartilage is avascular, molecular transport occurs primarily via diffusion or convection, and cartilage matrix structure and composition may affect diffusive transport. Because of the inhomogeneous compressive properties of articular cartilage, we hypothesized that compression would decrease macromolecular diffusivity and increase diffusional anisotropy in a site-specific manner that depends on local tissue strain. We used two fluorescence photobleaching methods, scanning microphotolysis and fluorescence imaging of continuous point photobleaching, to measure diffusion coefficients and diffusional anisotropy of 70 kDa dextran in cartilage during compression, and measured local tissue strain using texture correlation. For every 10% increase in normal strain, the fractional change in diffusivity decreased by 0.16 in all zones, and diffusional anisotropy increased 1.1-fold in the surface zone and 1.04-fold in the middle zone, and did not change in the deep zone. These results indicate that inhomogeneity in matrix structure and composition may significantly affect local diffusive transport in cartilage, particularly in response to mechanical loading. Our findings suggest that high strains in the surface zone significantly decrease diffusivity and increase anisotropy, which may decrease transport between cartilage and synovial fluid during compression.     

Toll-like receptors pathway in common variable immune deficiency (CVID) and X-linked agammaglobulinemia (XLA)

Common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA) are two major humoral immunodeficiencies, causing a high rate of early age mortality in children. In order to identifiy the possible factors involved in the pathogenesis of CVID and XLA, recent studies have focused on Toll-like receptors (TLRs) and demonstrate the defects in different TLR pathways in immune cells of CVID and XLA patients. Herein, we measured TLR-4 and TLR-9 RNA levels and consequently TNF-α and IFN-α production in peripheral blood mononuclear cells (PBMCs) of patients with CVID and XLA. Contrary to healthy individuals, TLR-9 expression was not significantly increased after ligand stimulation, whereas ligand-induced TLR-4 expression was not significantly different from that in healthy control PBMCs. Lipopolysaccharide (LPS)-stimulated TNF-α production was significantly reduced in patients compared to controls, whereas IFN-α production was increased in all groups after CpG stimulation without any significant inter-group difference. Our data suggest that defects in TLR-9 activated pathways may be a result of the decreased TLR-9 expression, although TLR-9 is not the only modulator of IFN-α production in these patients. On the other hand, impaired signaling in TLR-4 activated pathways which results in significant reduction in TNF-α production are not related to a defect in TLR-4 expression.     

Compression-induced changes in the shape and volume of the chondrocyte nucleus

Changes in cell shape and volume are believed to play a role in the process of mechanical signal transduction by chondrocytes in articular cartilage. One proposed pathway through which chondrocyte deformation may be transduced to an intracellular signal is through cytoskeletally mediated deformation of intracellular organelles, and more specifically, of the cell nucleus. In this study, confocal scanning laser microscopy was used to perform in situ three-dimensional morphometric analyses of the nuclei of viable condrocytes during controlled compression of articular cartilage explants from the canine patellofemoral groove. Unconfined compression of the tissue to a 15% surface-to-surface strain resulted in a significant decrease of chondrocyte height and volume by 14.7 ± 6.4 and 11.4 ± 8.4%, respectively, and of nuclear height and volume by 8.8 ± 6.2% and 9.8 ± 8.8%, respectively. Disruption of the actin cytoskeleton using cytochalasin D altered the relationship between matrix deformation and changes in nuclear height and shape, but not volume. The morphology and deformation behavior of the chondrocytes were not affected by cytochalasin treatment. These results suggest that the actin cytoskeleton plays an important role in the link between compression of the extracellular matrix and deformation of the chondrocyte nuclei and imply that chondrocytes and their nuclei undergo significant changes in shape and volume in vivo.     

A biomechanical role for perlecan in the pericellular matrix of articular cartilage

Chondrocytes are surrounded by a narrow pericellular matrix (PCM) that is biochemically, structurally, and biomechanically distinct from the bulk extracellular matrix (ECM) of articular cartilage. While the PCM is often defined by the presence of type VI collagen, other macromolecules such as perlecan, a heparan sulfate (HS) proteoglycan, are also exclusively localized to the PCM in normal cartilage and likely contribute to PCM structural integrity and biomechanical properties. Though perlecan is essential for normal cartilage development, its exact role in the PCM is unknown. The objective of this study was to determine the biomechanical role of perlecan in the articular cartilage PCM in situ and its potential as a defining factor of the PCM. To this end, atomic force microscopy (AFM) stiffness mapping was combined with dual immunofluorescence labeling of cryosectioned porcine cartilage samples for type VI collagen and perlecan. While there was no difference in overall PCM mechanical properties between type VI collagen- and perlecan-based definitions of the PCM, within the PCM, interior regions containing both type VI collagen and perlecan exhibited lower elastic moduli than more peripheral regions rich in type VI collagen alone. Enzymatic removal of HS chains from perlecan with heparinase III increased PCM elastic moduli both overall and locally in interior regions rich in both perlecan and type VI collagen. Heparinase III digestion had no effect on ECM elastic moduli. Our findings provide new evidence for perlecan as a defining factor in both the biochemical and biomechanical properties of the PCM.     

Regional differences in prostaglandin E2 and nitric oxide production in the knee meniscus in response to dynamic compression

Injury or loss of the knee meniscus is associated with altered joint stresses that lead to progressive joint degeneration. The goal of this study was to determine if dynamic mechanical compression influences the production of inflammatory mediators by meniscal cells. Dynamic compression increased prostaglandin E2 (PGE(2)) and nitric oxide (NO) production over a range of stress magnitudes (0.0125-0.5 MPa) in a manner that depended on stress magnitude and zone of tissue origin. Inner zone explants showed greater increases in PGE(2) and NO production as compared to outer zone explants. Meniscal tissue expressed NOS2 and NOS3 protein, but not NOS1. Mechanically induced NO production was blocked by NOS inhibitors, and the non-selective NOS inhibitor L-NMMA augmented PGE(2) production in the outer zone only. These findings suggest that the meniscus may serve as an intra-articular source of pro-inflammatory mediators, and that alterations in the magnitude or distribution of joint loading could significantly influence the production of these mediators in vivo.     

BRCA1 maps proximal to D17S579 on chromosome 17q21 by genetic analysis

Previous studies have demonstrated linkage between early-onset breast cancer and ovarian cancer and genetic markers on chromosome 17q21. These markers define the location of a gene (BRCA1) which appears to be inherited as an autosomal dominant susceptibility allele. We analyzed five families with multiple affected individuals for evidence of linkage to the BRCA1 region. Two of the five families appear to be linked to BRCA1. One apparently linked family contains critical recombinants, suggesting that the gene is proximal to the marker D17S579 (Mfd188). These findings are consistent with the maximum-likelihood position estimated by the Breast Cancer Linkage Consortium and with recombination events detected in other linked families. Linkage analysis was greatly aided by PCR-based analysis of paraffin-embedded normal breast tissue from deceased family members, demonstrating the feasibility and importance of this approach. One of the two families with evidence of linkage between breast cancer and genetic markers flanking BRCA1 represents the first such family of African-American descent to be reported in detail.     

Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells

Articular cartilage is an avascular connective tissue that exhibits little intrinsic capacity for repair. Articular cartilage exists in a reduced oxygen ( approximately 5%) environment in vivo; therefore, oxygen tension may be an important factor that regulates the metabolism of chondrocyte progenitors. A number of recent studies have developed tissue engineering approaches for promoting cartilage repair using undifferentiated progenitor cells seeded on biomaterial scaffolds, but little is known about how oxygen might influence these engineered tissues. Human adipose-derived adult stem (hADAS) cells isolated from the stroma of subcutaneous fat were suspended in alginate beads and cultured in control or chondrogenic media in either low oxygen (5%) or atmospheric oxygen tension (20%) for up to 14 days. Under chondrogenic conditions, low oxygen tension significantly inhibited the proliferation of hADAS cells, but induced a two-fold increase in the rate of protein synthesis and a three-fold increase in total collagen synthesis. Low oxygen tension also increased glycosaminoglycan synthesis at certain timepoints. Immunohistochemical analysis showed significant production of cartilage-associated matrix molecules, including collagen type II and chondroitin-4-sulfate. These findings suggest oxygen tension may play an important role in regulating the proliferation and metabolism of hADAS cells as they undergo chondrogenesis, and the exogenous control of oxygen tension may provide a means of increasing the overall accumulation of matrix macromolecules in tissue-engineered cartilage.