Comparative glycomics of connective tissue glycosaminoglycans.

Homeostasis of connective joint tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis.

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling.

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis.

Identification of cis and trans-acting transcriptional regulators in chondroinduced fibroblasts from the pre-phenotypic gene expression profile.

Cell differentiation is regulated via expression of successive sets of genes. In an in vitro model of chondrocyte differentiation, human dermal fibroblasts (hDFs) cultured in collagen sponges are induced to express cartilage matrix genes after 7 days' culture with demineralized bone powder (DBP). A shift in expression of many other genes occurs within 3 days, before chondroblast phenotypic genes are detectable.

Wnt influence on chondrocyte differentiation and cartilage function.

The Wnt signaling network regulates chondrocyte differentiation, proliferation, and maturation during embryonic limb development. In this review, we summarize studies of Wnt signaling during the chondrocyte life cycle in avian and mammalian systems, both before and after birth. Recent reports that implicate abnormal Wnt signaling as a contributing factor to pathogenic joint conditions are also discussed.

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells.

Spatial and temporal changes in the subcellular localization of the nuclear protein-tyrosine kinase, c-Fes.

Tyrosine phosphorylation has emerged as a mechanism to control cellular events in the nucleus. The c-Fes protein-tyrosine kinase is an important regulator of cell growth and differentiation in several cell types, and is found in the nucleus of hematopoietic cells. In this study, we showed nuclear localization of c-Fes in both hematopoietic (K562, TF-1, HEL, U937, and HL-60) and nonhematopoietic cell lines (293T, CaOv3, TfxH, MG-63, HeLa, DU-145) by immunofluorescence and confocal microscopy.

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes.

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans.

Demineralized bone promotes chondrocyte or osteoblast differentiation of human marrow stromal cells cultured in collagen sponges.

Demineralized bone implants have been used for many types of craniomaxillofacial, orthopedic, periodontal, and hand reconstruction procedures. In previous studies, we showed that demineralized bone powder (DBP) induces chondrogenesis of human dermal fibroblasts in a DBP/collagen sponge system that optimized interactions between particles of DBP and target cells in cell culture. In this study, we test the hypothesis that DBP promotes chondrogenesis or osteogenesis of human marrow stromal cells (hMSCs) in 3-D collagen sponge culture, depending upon the culture conditions.

Comparison of TGF-beta/BMP pathways signaled by demineralized bone powder and BMP-2 in human dermal fibroblasts.

Unlabelled: Demineralized bone induces chondrogenic differentiation of human dermal fibroblasts in vitro. Analyses of signaling gene expression showed that DBP and BMP-2 regulate common and distinct pathways. Although BMP-2 was originally isolated as a putative active factor in DBP, rhBMP-2 and DBP do not affect all the same genes or in the same ways.

Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts.

Objective: The Wnt family of secreted proteins, their receptors (Fzd proteins) and antagonists (secreted Fzd-related proteins, or Sfrp) regulate chondrocyte differentiation and chrondrogenesis during embryonic development. Here, the hypothesis that the Wnt regulatory network contributes to chondrocyte differentiation of post-natal cells was tested in an in vitro model of chondroinduction by demineralized bone powder (DBP).

Design: Human dermal fibroblasts (hDFs) were cultured in porous, three-dimensional (3D) collagen sponges with or without chondroinductive DBP.

Inferred functions of "novel" genes identified in fibroblasts chondroinduced by demineralized bone.

Little is known about the cellular mechanisms that control postnatal chondrocyte differentiation. As a first step towards identifying those mechanisms, gene expression shifts were characterized in an in vitro model of chondroinduction. In previous studies, several functional classes of genes (cytoskeletal and matrix elements, cell adhesion proteins, peptide growth factors, and signal transduction proteins) were found to be altered in human dermal fibroblasts (hDFs) cultured in porous collagen sponges with chondroinductive demineralized bone powder (DBP) for 3 days.

New chondrocyte genes discovered by representational difference analysis of chondroinduced human fibroblasts.

This report includes a review of the potential for gene expression analyses to provide new information for solving problems in skeletal repair and regeneration. It focuses on two approaches: high-throughput gene array methods and representational difference analysis (RDA). The principles underlying these methods are presented with experimental tutorials and some applications.

Altered expression of connective tissue genes in postnatal chondroinduced human dermal fibroblasts.

In a novel model for postnatal chondroinduction, normal human dermal fibroblasts (hDFs) cultured with demineralized bone powder (DBP) express chondrocyte features after 7 days. Representational difference analysis (RDA) prior to overt chondroblastogenesis (3 days) revealed altered expression of connective tissue genes (collagens, collagen receptors, and post-translational enzymes). Northern or RT-PCR analysis at 3, 7, 14, or 21 d showed different expression patterns for those genes.

Gene expression changes in an in vitro model of chondroinduction: a comparison of two methods.

There are many useful technologies to describe patterns of gene expression that occur during tissue repair and regeneration. Results from different methods used in one experimental setting are not often compared. In this case study of chondrogenesis, we compare two methods to identify differentially expressed genes, representational difference analysis and targeted macroarray analysis, as a model for investigating genes that may be relevant to tissue repair.

Nerve growth factor is expressed in rat femoral vein.

Purpose: Entubulization is a well known method of nerve repair for defects too large to be reconstructed by direct suturing without tension. Vein grafts and alloplastic tubes have been used for entubulization in peripheral and cranial nerves, but the mechanism by which they promote healing is poorly understood. The overall hypothesis of this laboratory is that nerve growth factor (NGF) plays an important role in nerve regeneration after entubulization with a vein graft.