Incorporation and immunogenicity of cleaned bovine bone in a sheep model.

This study was conducted to determine if a novel cleaning process could extract antigenic material from bovine bone thereby improving incorporation. Cleaned bovine xenograft, untreated bovine xenograft and sheep allograft were implanted into the tibia of mature sheep for 12 and 24 weeks. Inflammation, bone integration and immunological reactions were evaluated via standardized assays. Cleaned bovine bone dowels induced significantly lower inflammatory responses (p < 50.05) when compared to traditionally processed xenograft. Bone integration, measured by in situ biomechanics, was not different between cleaned bovine bone and allograft controls (p = 0.96). A transient antibody response was observed for non-treated xenografts although this response abated by 3 months.

Out-of-equilibrium dynamics in the cytoskeleton of the living cell.

We report here measurements of rheological properties of the human airway smooth muscle cell using forced nanoscale motions of Arg-Gly-Asp RGD-coated microbeads tightly bound to the cytoskeleton. With changes of forcing amplitude, the storage modulus showed small but systematic nonlinearities, especially after treatment with a contractile agonist. In a dose-dependent manner, a large oscillatory shear applied from a few seconds up to 400 s caused the cytoskeleton matrix to soften, a behavior comparable to physical rejuvenation observed in certain inert soft materials; the stiffness remained constant for as long as the large oscillatory shear was maintained, but suddenly fell with shear cessation. Stiffness then followed a slow scale-free recovery, a phenomenon comparable to physical aging. However, acetylated low-density lipoprotein acLDL-coated microbeads, which connect mainly to scavenger receptors, did not show similar out-of-equilibrium behaviors. Taken together, these data demonstrate in the cytoskeleton of the living cell behaviors with all the same signatures as that of soft inert condensed systems. This unexpected intersection of condensed matter physics and cytoskeletal biology suggests that trapping, intermittency, and approach to kinetic arrest represent central mesoscale features linking underlying molecular events to integrative cellular functions.

Role of heat shock protein 27 in cytoskeletal remodeling of the airway smooth muscle cell.

Remodeling of the airway smooth muscle (ASM) cell has been proposed to play an important role in airway hyperresponsiveness. Using a functional assay, we have assessed remodeling of the cultured rat ASM cell and the role of heat shock protein (HSP) 27 in that process. To probe remodeling dynamics, we measured spontaneous motions of an individual Arg-Gly-Asp-coated microbead that was anchored to the cytoskeleton. We reasoned that the bead could not move unless the microstructure to which it is attached rearranged; if so, then its mean square displacement (MSD) would report ongoing internal reorganizations over time. Each bead displayed a random, superdiffusive motion; MSD increased with time as approximately t(1.7), whereas an exponent of unity would be expected for a simple passive diffusion. Increasing concentrations of cytochalasin-D or latrunculin-A caused marked increases in the MSD, whereas colchicine did not. Treatments with PDGF or IL-1beta, but not transforming growth factor-beta, caused decreases in the MSD, the extent of which rank-ordered with the relative potency of these agents in eliciting the phosphorylation of HSP27. The chemical stressors anisomycin and arsenite each increased the levels of HSP27 phosphorylation and, at the same time, decreased bead motions. In particular, arsenite prevented and even reversed the effects of cytochalasin-D on bead motions. Finally, ASM cells overexpressing phospho-mimicking human HSP27, but not wild-type or phosphorylation-deficient HSP27, exhibited decreases in bead motions that were comparable to the arsenite response. Taken together, these results show that phosphorylated HSP27 favors reduced bead motions that are probably due to stabilization of the actin cytoskeleton.

Demineralized bone matrix as an osteoinductive biomaterial and in vitro predictors of its biological potential.

The osteoinductivity of demineralized bone matrix (DBM) varies from donor to donor as a result of varying levels of multiple growth factors, matrix integrity, and artifacts from material processing. Many in vitro assays are currently used for screening the osteoinductivity of DBM. The objectives of this study were to determine the correlation of specific growth factors and in vitro mitotic stimulation to in vivo ectopic bone formation capacity with a large number of DBM samples. Samples were assayed using ELISA methods for BMP-2/4 and TGF-beta1 (n = 304) and cell proliferation using SAOS-2 osteoblasts (n = 239). All samples were then implanted intramuscularly in the abdomen of nude rats. All in vitro assays showed significant variability for any particular level of ostoinductivity determined by in vivo model. A significant, but only very weak, positive correlation to in vivo results was found for TGF-beta1 (r(2) = 0.016), BMP 2/4 (r(2) = 0.065), and SAOS-2 cell proliferation (r(2) = 0.053). The results of this study amplify the notion that a multitude of factors and their relative interplay, rather than a single factor are likely to determine the potency of any particular lot of DBM.

Influence of donor age on the biomechanical and biochemical properties of human meniscal allografts.

BACKGROUND: Although the use of meniscal allografts to replace severely damaged or absent menisci is commonplace, little is known about the effects of donor age on the biochemical and biomechanical properties of human menisci. HYPOTHESIS: The mechanical and biochemical properties of human medial and lateral menisci from donors less than 45 years of age do not vary with donor age. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-three lateral and 25 medial menisci from 34 donors (26 male and 8 female) ranging from 15 to 44 years of age were harvested and immediately stored at -80 degrees C. The outer third of each meniscus was subjected to static and dynamic tensile analysis. In addition, the biochemical composition (collagen, proteoglycan, and water content) of these samples was analyzed. RESULTS: There was no correlation between donor age and static tensile stiffness for either the lateral (R(2) = .003) or medial (R(2) = .002) meniscus. Likewise, there was no correlation between donor age and dynamic tensile modulus for either the lateral or medial meniscus. Although there was a weak, positive correlation between water content and age in both lateral (R(2) = .22) and medial (R(2) = .25) menisci, there was no effect of age on collagen or proteoglycan content. There were no differences (P > .05) between female and male menisci in any of the measured biomechanical or biochemical parameters tested. CONCLUSION: The tensile properties, as well as the collagen and proteoglycan content, of menisci from donors less than 45 years of age were not age dependent. CLINICAL RELEVANCE: The age of the donor does not appear to affect the initial tensile properties of menisci from donors less than 45 years of age.

Directional memory and caged dynamics in cytoskeletal remodelling.

We report directional memory of spontaneous nanoscale displacements of an individual bead firmly anchored to the cytoskeleton of a living cell. A novel method of analysis shows that for shorter time intervals cytoskeletal displacements are antipersistent and thus provides direct evidence in a living cell of molecular trapping and caged dynamics. At longer time intervals displacements are persistent. The transition from antipersistence to persistence is indicative of a time-scale for cage rearrangements and is found to depend upon energy release due to ATP hydrolysis and proximity to a glass transition. Anomalous diffusion is known to imply memory, but we show here that memory is attributed to direction rather than step size. As such, these data are the first to provide a molecular-scale physical picture describing the cytoskeletal remodelling process and its rate of progression.

Cytoskeleton dynamics: fluctuations within the network.

Out-of-equilibrium systems, such as the dynamics of a living cytoskeleton (CSK), are inherently noisy with fluctuations arising from the stochastic nature of the underlying biochemical and molecular events. Recently, such fluctuations within the cell were characterized by observing spontaneous nano-scale motions of an RGD-coated microbead bound to the cell surface [Bursac et al., Nat. Mater. 4 (2005) 557-561]. While these reported anomalous bead motions represent a molecular level reorganization (remodeling) of microstructures in contact with the bead, a precise nature of these cytoskeletal constituents and forces that drive their remodeling dynamics are largely unclear. Here, we focused upon spontaneous motions of an RGD-coated bead and, in particular, assessed to what extent these motions are attributable to (i) bulk cell movement (cell crawling), (ii) dynamics of focal adhesions, (iii) dynamics of lipid membrane, and/or (iv) dynamics of the underlying actin CSK driven by myosin motors.

Filamin-a and rheological properties of cultured melanoma cells.

Here we report the rheological properties of cultured hsFLNa (filamin-A)-expressing (FIL+) and hsFLNa-deficient (FIL-) melanoma cells. Using magnetic twisting cytometry over a wide range of probing frequencies, and targeting either cortical or deeper cytoskeletal structures, we found that differences in stiffness of FIL+ versus FIL- cells were remarkably small. When probed through deep cytoskeletal structures, FIL+ cells were, at most, 30% stiffer than FIL- cells, whereas when probed through more peripheral cytoskeletal structures FIL- cells were not different except at very high frequencies. The loss tangent, expressed as an effective cytoskeletal temperature, was systematically greater in FIL- than FIL+ cells, but these differences were small and showed that the FIL+ cells were only slightly closer to a solidlike state. To quantify cytoskeletal remodeling, we measured spontaneous motions of beads bound to cortical cytoskeletal structures and found no difference in FIL+ versus FIL- cells. Although mechanical differences between FIL+ and FIL- cells were evident both in cortical and deeper structures, these differences were far smaller than expected based on measurements of the rheology of purified actin-filamin solutions. These findings do not rule out an important contribution of filamin to the mechanical properties of the cortical cytoskeleton, but suggest that effects of filamin in the cortex are not exerted on the length scale of the probe used here. These findings would appear to rule out any important contribution of filamin to the bulk mechanical properties of the cytoplasm, however. Although filamin is present in the cytoplasm, it may be inactive, its mechanical effects may be small compared with other crosslinkers, or mechanical properties of the matrix may be dominated by an overriding role of cytoskeletal prestress.

Cytoskeletal remodelling and slow dynamics in the living cell.

The cytoskeleton (CSK) is a crowded network of structural proteins that stabilizes cell shape and drives cell motions. Recent studies on the dynamics of the CSK have established that a wide variety of cell types exhibit rheology in which responses are not tied to any particular relaxation times and are thus scale-free. Scale-free rheology is often found in a class of materials called soft glasses, but not all materials expressing scale-free rheology are glassy (see plastics, wood, concrete or some metals for example). As such, the extent to which dynamics of the CSK might be regarded as glassy remained an open question. Here we report both forced and spontaneous motions of microbeads tightly bound to the CSK of human muscle cells. Large oscillatory shear fluidized the CSK matrix, which was followed by slow scale-free recovery of rheological properties (aging). Spontaneous bead motions were subdiffusive at short times but superdiffusive at longer times; intermittent motions reflecting nanoscale CSK rearrangements depended on both the approach to kinetic arrest and energy release due to ATP hydrolysis. Aging, intermittency, and approach to kinetic arrest establish a striking analogy between the behaviour of the living CSK and that of inert non-equilibrium systems, including soft glasses, but with important differences that are highly ATP-dependent. These mesoscale dynamics link integrative CSK functions to underlying molecular events, and represent an important intersection of topical issues in condensed matter physics and systems biology.