Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2 = 0.47, p < 0.001) and GAG content (r2 = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2 = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

An evaluation of alternative media for pebble matrix filtration using clay balls and recycled crushed glass

Protecting slow sand filters (SSFs) from high-turbidity waters by pretreatment using pebble matrix filtration (PMF) has previously been studied in the laboratory at University College London, followed by pilot field trials in Papua New Guinea and Serbia. The first full-scale PMF plant was completed at a water-treatment plant in Sri Lanka in 2008, and during its construction, problems were encountered in sourcing the required size of pebbles and sand as filter media. Because sourcing of uniform-sized pebbles may be problematic in many countries, the performance of alternative media has been investigated for the sustainability of the PMF system. Hand-formed clay balls made at a 100-yearold brick factory in the United Kingdom appear to have satisfied the role of pebbles, and a laboratory filter column was operated by using these clay balls together with recycled crushed glass as an alternative to sand media in the PMF. Results showed that in countries where uniform-sized pebbles are difficult to obtain, clay balls are an effective and feasible alternative to natural pebbles. Also, recycled crushed glass performed as well as or better than silica sand as an alternative fine media in the clarification process, although cleaning by drainage was more effective with sand media. In the tested filtration velocity range of ð0:72–1:33Þ m=h and inlet turbidity range of (78–589) NTU, both sand and glass produced above 95% removal efficiencies. The head loss development during clogging was about 30% higher in sand than in glass media.

Learning the kernel matrix with semidefinite programming

Kernel-based learning algorithms work by embedding the data into a Euclidean space, and then searching for linear relations among the embedded data points. The embedding is performed implicitly, by specifying the inner products between each pair of points in the embedding space. This information is contained in the so-called kernel matrix, a symmetric and positive semidefinite matrix that encodes the relative positions of all points. Specifying this matrix amounts to specifying the geometry of the embedding space and inducing a notion of similarity in the input space - classical model selection problems in machine learning. In this paper we show how the kernel matrix can be learned from data via semidefinite programming (SDP) techniques. When applied to a kernel matrix associated with both training and test data this gives a powerful transductive algorithm -using the labeled part of the data one can learn an embedding also for the unlabeled part. The similarity between test points is inferred from training points and their labels. Importantly, these learning problems are convex, so we obtain a method for learning both the model class and the function without local minima. Furthermore, this approach leads directly to a convex method for learning the 2-norm soft margin parameter in support vector machines, solving an important open problem.

Matrix regularization techniques for online multitask learning

In this paper we examine the problem of prediction with expert advice in a setup where the learner is presented with a sequence of examples coming from different tasks. In order for the learner to be able to benefit from performing multiple tasks simultaneously, we make assumptions of task relatedness by constraining the comparator to use a lesser number of best experts than the number of tasks. We show how this corresponds naturally to learning under spectral or structural matrix constraints, and propose regularization techniques to enforce the constraints. The regularization techniques proposed here are interesting in their own right and multitask learning is just one application for the ideas. A theoretical analysis of one such regularizer is performed, and a regret bound that shows benefits of this setup is reported.

Learning the Kernel Matrix with Semi-Definite Programming

Kernel-based learning algorithms work by embedding the data into a Euclidean space, and then searching for linear relations among the embedded data points. The embedding is performed implicitly, by specifying the inner products between each pair of points in the embedding space. This information is contained in the so-called kernel matrix, a symmetric and positive definite matrix that encodes the relative positions of all points. Specifying this matrix amounts to specifying the geometry of the embedding space and inducing a notion of similarity in the input space -- classical model selection problems in machine learning. In this paper we show how the kernel matrix can be learned from data via semi-definite programming (SDP) techniques. When applied to a kernel matrix associated with both training and test data this gives a powerful transductive algorithm -- using the labelled part of the data one can learn an embedding also for the unlabelled part. The similarity between test points is inferred from training points and their labels. Importantly, these learning problems are convex, so we obtain a method for learning both the model class and the function without local minima. Furthermore, this approach leads directly to a convex method to learn the 2-norm soft margin parameter in support vector machines, solving another important open problem. Finally, the novel approach presented in the paper is supported by positive empirical results.

Extracellular matrix of the human cyclic corpus luteum

Extracellular matrix regulates many cellular processes likely to be important for development and regression of corpora lutea. Therefore, we identified the types and components of the extracellular matrix of the human corpus luteum at different stages of the menstrual cycle. Two different types of extracellular matrix were identified by electron microscopy; subendothelial basal laminas and an interstitial matrix located as aggregates at irregular intervals between the non-vascular cells. No basal laminas were associated with luteal cells. At all stages, collagen type IV α1 and laminins α5, β2 and γ1 were localized by immunohistochemistry to subendothelial basal laminas, and collagen type IV α1 and laminins α2, α5, β1 and β2 localized in the interstitial matrix. Laminin α4 and β1 chains occurred in the subendothelial basal lamina from mid-luteal stage to regression; at earlier stages, a punctate pattern of staining was observed. Therefore, human luteal subendothelial basal laminas potentially contain laminin 11 during early luteal development and, additionally, laminins 8, 9 and 10 at the mid-luteal phase. Laminin α1 and α3 chains were not detected in corpora lutea. Versican localized to the connective tissue extremities of the corpus luteum. Thus, during the formation of the human corpus luteum, remodelling of extracellular matrix does not result in basal laminas as present in the adrenal cortex or ovarian follicle. Instead, novel aggregates of interstitial matrix of collagen and laminin are deposited within the luteal parenchyma, and it remains to be seen whether this matrix is important for maintaining the luteal cell phenotype.

Homodimerization controls the fibroblast growth factor 9 subfamily's receptor binding and heparan sulfate-dependent diffusion in the extracellular matrix

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20's receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.