Alteration of matrix glycosaminoglycans diminishes articular chondrocytes' response to a canonical Wnt signal.

OBJECTIVE: Although Wnt signaling is a key regulator of the chondrocyte life cycle during embryonic development, little is known about Wnt activity in articular cartilage. Recent studies have suggested an association between excess signaling through the canonical Wnt pathway and osteoarthritis (OA). Genetic and in vitro studies with Drosophila have shown that signaling by the orthologous protein, Wingless (Wg), is regulated by glycosaminoglycans (GAGs) found at the cell surface. The objective of this study was to determine whether alteration in GAG sulfation or matrix content, such as that occurs in OA cartilage, would affect articular chondrocytes' response to a canonical Wnt stimulus. METHODS: Cells were isolated from shoulder joints of young calves (bovine articular chondrocytes, bACs) and from human cartilage (human articular chondrocytes, hACs) discarded during total knee replacement for OA. Conditioned media from a cell line that is stably transfected with Wnt3a was used as a source of Wnt protein that activates the canonical signaling pathway. Conditioned media from the parental cell line was used as a control. beta-catenin levels were measured by immunoblot. In some experiments, chondrocyte cultures were treated with sodium chlorate (NaClO3) to inhibit GAG sulfation, or with chondroitinase ABC (ChABC) to digest chondroitin sulfate (CS) in the matrix. RESULTS: Cultured bACs showed low steady-state levels of beta-catenin that increased upon stimulation with Wnt3a. A decrease in either GAG sulfation or CS content diminished bACs' response to Wnt3a (approximately 40% and 37% of control, respectively). Similar effects on the response to Wnt3a via beta-catenin were observed for cultured hACs with undersulfation of GAGs (16% of control) and decreased CS content (20% of control). CONCLUSION: This study demonstrates that articular chondrocytes respond to canonical Wnt stimulation, and that reduced sulfation or CS content diminishes that response.

In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha.

OBJECTIVES: Cartilage is avascular and relatively homogeneous, making it an attractive tissue for in vitro histogenesis and surgical use in patients. We developed novel platform technologies in order to define the requirements for optimal in vitro chondrogenesis by isolated cells. In this series of studies, we tested alternatives to fetal bovine serum (FBS) and the effects of growth factors on formation of cartilage in 3D porous collagen sponges. DESIGN: We used porous collagen sponges to assess the effects of serum substitutes and exogenous TGF-beta1 and IL-1alpha on chondrocytes (bovine articular chondrocytes, bACs) and on chondroinduced human dermal fibroblasts (hDFs). We determined the effects of low concentrations of FBS and two serum substitutes, Nutridoma and ITS(+3), on cellularity and matrix production. After culture for intervals, sponges were harvested for histological and biochemical measurement of cartilage-specific chondroitin 4-sulfate proteoglycan (C 4-S PG). RESULTS: Cultured bACs showed equivalent growth in Nutridoma (1%) and 10% FBS. Both TGF-beta1 and IL-1alpha significantly stimulated accumulation of C 4-S PG by bACs in 3D porous collagen sponges. Many endogenous growth factors were upregulated in hDFs cultured with chondroinductive DBP. Addition of TGF-beta1 and IL-1alpha for 11 days significantly stimulated accumulation of C 4-S PG by hDFs cultured in DMEM with 1% Nutridoma. CONCLUSION: Porous collagen sponges are supportive of chondrogenesis and of chondroinduction by DBP. Optimization of serum-free culture conditions, including growth factors, matrix components, and mechanical stimuli will expedite translation to wider clinical applications. Use of autogenous dermal fibroblasts pre-cultured with DBP and induced to chondrocytes offers an alternative to autogenous chondrocytes.

Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee.

This study assessed the feasibility of a devitalized knee as a scaffold for an engineered chimeric joint. Embryonic chick knees (19 days old), devitalized by lyophilization or multiple freeze-thaw cycles, were tested as scaffolds for repopulation with bovine articular chondrocytes (bACs). bACs were seeded into porous three-dimensional collagen sponges and were cultured for 1 day before fabrication of chimeric constructs. A pair of cell-seeded sponges was inserted into the joint space to contact preshaved articular surfaces. In some constructs, a sterile membrane of expanded polytetrafluoroethylene (ePTFE) was inserted between the collagen sponges. Histologic analysis showed that at 1 week, sponges with bACs were adherent to the shaved articular surfaces of the joint with accumulation of metachromatic extracellular matrix. Penetration of bACs and neomatrix into the devitalized matrix appeared to begin in preexistent epiphyseal canals and was observed to some extent in all specimens. Membranes of ePTFE maintained a joint space at 2 and 3 weeks, whereas there was fusion across the two sponges in many specimens lacking the membrane. Gene expression analysis demonstrated that lyophilization, but not multiple freeze-thaw cycles, completely devitalized the chick knees. These studies identified several design parameters crucial for successful engineering of a chimeric joint.

IGF-I, TGF-beta, and BMP-4 are expressed during distraction osteogenesis of the pig mandible.

The mechanisms that regulate bone formation during distraction osteogenesis (DO) are not completely understood. Our hypothesis is that local cytokines that stimulate osteoblast activity are potential regulators of this process. The purpose of this study was to determine gene expression of insulin-like growth factor I (IGF-I), transforming growth factor-beta (TGF-beta), and bone morphogenetic protein 4 (BMP-4) in distracted wounds. A semiburied, rigid distraction device was placed across an osteotomy at the right mandibular angle in 9 Yucatan minipigs. Distraction was begun immediately at a rate of 1 mm/day. The animals were sacrificed after 4 and 7 days of distraction, and after 7 days of distraction plus 4 days of neutral fixation. Excised wound tissues were processed for histologic and gene expression analyses. Competitive reverse-transcription polymerase chain reaction (RT-PCR) assays were developed and validated for porcine genes. Histologic analysis showed membranous ossification within the DO wound. Gene expression of IGF-I, TGF-beta and BMP-4 was detected during distraction and neutral fixation. These results show that gene expression analyses can be performed in a large animal model of mandibular DO. As the pig mandible closely resembles that of the human in morphology and physiology, this is an important step toward characterization of the early molecular events in the DO wound.

Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds.

Hyaluronan (HA) is a component of cartilage matrix with known effects on chondrocytes. We tested the effects of adding HA to 3-dimensional (3-D) collagen. sponges on chondrocyte function in vitro. Bovine articular chondrocytes isolated by collagenase digestion were injected into either collagen or HA/collagen scaffolds comprising different amounts of HA (2, 5, 10, and 14% w/w). Expression of aggrecan and type II collagen genes was measured by gene-specific quantitative competitive reverse transcriptase-polymerase chain reactions, and the extracellular matrix was estimated by histomorphometrical analyses. After 7-day culture, the chondrocytes in 2% (w/w) HA sponges expressed fourfold more mRNA transcripts for type II collagen (p = 0.002) and twofold more mRNA transcripts for aggrecan (p = 0.022) than in control collagen sponges. Furthermore, there was 45% more extracellular matrix in 2% (w/w) HA sponges and 43% less matrix in the 10% (w/w) HA sponges compared with plain collagen sponges (p > 0.05). In sum, a small amount of HA in 3-D collagen scaffolds enhanced chondrogenesis, but a greater amount was inhibitory. This 3-D system represents a novel tool to identify mechanisms by which extracellular matrix molecules influence chondrocyte function. Further, these results show the potential for modifying scaffolds to improve production of engineered cartilage for in vivo applications.

Early shifts in gene expression during chondroinduction of human dermal fibroblasts.

Treatment options for damaged articular cartilage are limited because of that tissue's poor capacity for repair. Possible approaches to this problem are to stimulate cartilage matrix production in situ or to engineer replacement tissue. Both of these approaches would benefit from a detailed understanding of the molecular mechanisms of chondroblast differentiation. In previous studies, we described a novel in vitro model of postnatal chondroblast differentiation. That model of induced chondrogenesis was used to test the hypothesis that cellular interactions with demineralized bone powder (DBP) would induce specific, early shifts in gene expression, prior to the expression of cartilage matrix genes. Differentially expressed genes were identified by representational difference analysis of human dermal fibroblasts cultured for 3 days with DBP in three-dimensional collagen sponges. Genes that were upregulated by DBP comprised several functional classes, including cytoskeletal elements, protein synthesis and trafficking, and transcriptional regulation. Kinetic analysis of gene expression over 21 days showed that vigilin was transiently upregulated on day 3. In contrast, expression of cartilage signature genes continued to increase. These results are an important step toward complete characterization of the mechanisms by which DBP induces chondroblastic differentiation in postnatal cells.

Proliferation of masseter myocytes after distraction osteogenesis of the porcine mandible.

PURPOSE: Long-term success of distraction osteogenesis depends on the ability of the surrounding soft tissues to tolerate distraction forces and to adapt to the resulting increase in skeletal length and volume. The hypothesis tested in this study was that mandibular elongation by distraction induces myocyte proliferation. MATERIALS AND METHODS: Unilateral mandibular angle osteotomies were performed in 16 Yucatan minipigs. The hemimandibles were lengthened using semiburied distraction devices (Synthes Maxillofacial, Paoli, PA) with 0-day latency, 1, 2, or 4 mm/d distraction rates, and a neutral fixation period of twice the gap size in days. In 2 additional animals, the dissection and osteotomies were performed, and distraction devices were placed without activation (sham control). At the end of neutral fixation, tissues were taken from masseter muscle overlying the osteotomy and the equivalent region on the unoperated side (contralateral control). Proliferation of myocytes was estimated using immunohistochemical localization with antibodies against proliferating cell nuclear antigen (PCNA). RESULTS: Muscle overlying the distracted mandible showed 6-fold more PCNA-positive myocytes (16.8% +/- 11.3%) than the contralateral control side (2.8% +/- 1.1%, P < .0001). In the 2 sham-control animals, there was a low index of PCNA-positive myocytes on both the osteotomy (2%, 5%) and the contralateral sides (1%, 2%). CONCLUSIONS: The results of this study suggest that distraction of the porcine mandible by the protocol described induces myocyte proliferation in the masseter muscle. A proliferative response may contribute to improved long-term stability of mandibular expansion by distraction osteogenesis.

Analysis of Fes kinase activity in myeloid cell growth and differentiation.

Fes is a nonreceptor protein tyrosine kinase that has been implicated in a variety of cytokine signal transduction pathways, as well as differentiation of myeloid cells. To address the role of Fes in these processes, we overexpressed a kinase-defective Fes protein in the factor-dependent cell-lines, TF-1 and 32D. Proliferative responses to GM-CSF and interleukin 3, and the induction of differentiation by G-CSF were not altered by expression of the kinase mutant Fes protein, indicating that Fes kinase activity is not critical for these biological events in these cell lines.

Role of c-Fes in normal and neoplastic hematopoiesis.

The study of oncogenes has provided numerous insights, not only into the mechanisms by which growth regulation becomes uncontrolled in cancer cells, but also into signal transduction processes which regulate the orderly proliferation and maturation of cells. c-fes/fps is a cellular oncogene which has been transduced frequently by mammalian and avian retroviruses. There are several features about Fes which suggest it may play a unique role in myeloid cell growth and differentiation. While it contains a tyrosine kinase and SH2 domain, there is no SH3 domain or carboxy terminal regulatory phosphotyrosine such as found in the Src family of kinases. Fes has a unique N-terminal domain of over 400 amino acids of unknown function. It has been implicated in signaling by a variety of hematopoietic growth factors, and is predominantly a nuclear protein.

Human c-FES is a nuclear tyrosine kinase.

FES is a non-receptor protein tyrosine kinase expressed in hematopoietic progenitors and differentiated myeloid cells. It has recently been implicated in granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and erythropoietin signal transduction. To better understand the role played by FES in normal and neoplastic hematopoiesis, we used cell fractionation techniques to examine the subcellular localization of FES in myeloid cells and cell lines. FES was observed in the nuclear, granular and plasma membrane fractions of primary human neutrophils and the myeloid leukemia cell line, HL-60. The nuclear localization was confirmed by immunocytochemistry of neutrophils.

5' upstream sequence and genomic structure of the human primary response gene, EGR-1/TIS8.

EGR-1/TIS8 is a primary response gene that encodes a zinc finger containing protein and is induced in a number of cell types by a variety of ligands. We have isolated and mapped the human EGR-1/TIS8 gene and sequenced the 5' upstream flanking region. A 'TATA' homology and several putative regulatory elements, including two Sp1 sites, five serum response-like elements, two cAMP response-like elements, an EGR-1 binding site (EBS), and a tetra-decanoyl phorbol acetate (TPA)-responsive element have been identified within 700 nucleotides of the upstream region. We demonstrated that a 500-base pair fragment, which includes several of these possible regulatory sequences, is functional and responsive to TPA in transient transfection assays. A further understanding of the regulation and function of human EGR-1/TIS8 gene expression may provide insight into the mechanisms that control normal and neoplastic proliferation of human cells.