Paul A. Lucas

Chemotactic response of osteoblast-like cells to transforming growth factor beta

Transforming growth factor beta (TGF-beta) was tested for its ability to stimulate a chemotactic response in two clonal rat osteosarcoma (ROS) cell lines, 17/2 and 25/1. TGF-beta stimulated dose-dependent chemotaxis in both cell lines. In serum-containing media, maximal response was seen at a concentration of 500 fg (10(-15)g)/mL for the ROS 17/2 cells and 25 fg/mL for the ROS 25/1 cells. In serum-free media, the maximal chemotactic response to TGF-beta occurred at 5 fg/mL for both the ROS 17/2 and 25/1 cells. TGF-beta was not mitogenic at these dosages. The results indicate that TGF-beta could act as a chemoattractant for osteogenic cells in both demineralized bone matrix induced osteogenesis and in normal bone remodeling.

Inhibition of epidural scar formation after lumbar laminectomy in the rat

Study Design An animal model of laminectomy in rats was used to study scar tissue formation around the spinal cord. Dexamethasone, in controlled-release form, was tested in this system for its ability to decrease fibrous tissue formation. Objectives The results were evaluated to determine whether dexamethasone in a biodegradable controlledrelease vehicle could be used to limit scar tissue formation around the spinal cord after laminectomy Summary of Background Data Steroids can delay the formation of scar tissue. Continued treatment with dexamethasone results in various unacceptable side effects. Use of biodegradable controlled-release vehicles to deliver drugs may allow for prolonged low-dose treatment, concentrated at the surgical site, thereby avoiding side effects. Methods Forty-four Sprague Dawley rats underwent laminectomies and were treated with dexamethasone in one of two controlled-release vehicles or with vehicle alone. After 4 weeks, the rats were killed and histologic sections prepared from the spines were examined and graded by a pathologist. In addition, the dexamethasone preparations were introduced into Hunt-Schilling wound chambers, which were implanted in rats. Four weeks after implantation, the wound chambers were removed and the tissue inside was assayed for DNA and protein content. Results Dexamethasone acetate (Decadron, MSD, West Point, PA) significantly reduced the density of the scar tissue undermining the laminas. Steroids embedded in polymer did not change the scar formation in the back, but did decrease protein and DNA values in wound chamber tissues. Conclusions Long-term release of small amounts of steroid from the polymer poly-carboxy-phenoxypropane does not appear to reduce scar at laminectomy sites but does decrease the protein:DNA ratio in wound chambers. In contrast, Decadron does not significantly alter the biochemistry of wound chamber tissue but does reduce scar in the back.

Chemotactic response of embryonic limb bud mesenchymal cells and muscle-derived fibroblasts to transforming growth factor-β

Transforming growth factor beta (TGF-beta) was tested for its ability to stimulate a chemotactic response in Stage 24 embryonic chick limb bud mesenchymal cells and muscle-derived fibroblasts. TGF-beta stimulated dose-dependent chemotaxis in both cell populations. Maximal chemotaxis was achieved with a concentration of 5 ng/ml for limb bud cells and as low as 15 pg/ml for muscle-derived fibroblasts. TGF-beta was not chemokinetic at these levels. Several other proteins found in bone, namely fibronectin, type I collagen, and osteonectin, were not chemotactic. However, both Bone Gla-protein and basic-FGF were found to be chemotactic but less effective than TGF-beta. Comparison with extracts of adult bone indicates that while TGF-beta is a potent chemoattractant, it does not account for all the chemotactic activity found in adult bone.

Isolation of embryonic chick myosatellite and pluripotent stem cells

The current study outlines the isolation and culture of two populations of cells derived from Day 11 embryonic chick leg muscle and associated connective tissues. The two populations consisted of myogenic lineage-committed stem cells (myosatellite stem cells) and lineage-uncommitted stem cells (pluripotent stem cells). After long-term culture the lineage-uncommitted stem cell population displayed differentiated phenotypes suggestive of the following adult tissues, fibroblasts, muscle, fat, cartilage, and bone.

Cryopreservation of embryonic chick myogenic lineage-committed stem cells

Undifferentiated embryonic chick stem cells provide a good in vitro test system to examine the effects of recombinant growth factors on the resultant phenotypic expression of these cells. One of the major difficulties in using freshly isolated cells is variation in the proportion of stem cells recovered from different cell harvests. Variation is of particular concern when multiple cell harvests are necessary to provide cells for assaying a single growth factor. As an attempt to minimize this variation, we have examined the efficacy of cryopreserving myogenic lineage-committed stem cells derived from embryonic chick leg muscle. Percent recovery, viability, differentiation morphology, and cellular proliferation rates were compared between sister cultures of freshly isolated and cryopreserved stem cells. Embryonic chick stem cells retained their capacity to differentiate myogenically in culture when slowly frozen in and recovered from 7.5% dimethyl sulfoxide medium supplemented with horse serum and stage-specific embryo extract.

Enzyme-linked immuno-culture assay

The current study outlines a procedure, designated enzyme-linked immuno-culture assay (ELICA), that will measure phenotypic expression of cultured cells in small plate assays. Given standard curves for phenotypic expression markers and in situ DNA analysis, this procedure will quantitate (to nanogram levels) intracellular, cell surface, or extracellular phenotypic expression markers; visualize the location of the markers; and determine DNA content, all within the same well of a 24- or 96-well tissue culture plate.

Partial isolation and characterization of a chemotactic factor from adult bovine bone for mesenchymal cells

Demineralized adult bone matrix has the capacity to initiate de novo ectopic endochondral bone formation 2-3 weeks following intramuscular implantation into suitable hosts. An early step in this process is the migration of mesenchymal cells to the implant site; these cells later differentiate into cartilage and bone. Adult bone has been shown to contain a number of bioactive factors, such as chemotactic factors for various cell types, including osteoblasts. We have used embryonic chick limb bud mesenchymal cells to construct an in vitro assay for testing chemotactic activity derived from bone matrix extracts. With a modified Boyden chamber, water-soluble components from a 4 M guanidinium chloride extract of demineralized adult bovine bone matrix were found to stimulate the directional migration of these chick embryonic limb bud mesenchymal cells as well as embryonic muscle-derived fibroblasts and cells derived from embryonic skin. The chemotactic activity was destroyed by treatment with heat (100 degrees C) or trypsin. Partial purification by molecular sieve chromatography suggested that the chemotactic factor(s) has a molecular weight of between 50,000 and 90,000. This factor can be separated from bone matrix-derived chondrogenic stimulating activity by either ion exchange or molecular sieve chromatography. These observations confirm that bone matrix contains a chemoattractant for mesenchymal cells that may be important for in vivo recruitment of cells as part of the process of ectopic bone formation or in cases of bone repair.

Recombinant human bone morphogenetic proteins-2 and -4 induce several mesenchymal phenotypes in culture

Bone morphogenetic protein has previously been shown to induce the formation of cartilage and bone in vivo. We have isolated a population of mesenchymal stem cells from rat skeletal muscle capable of forming multiple mesodermal morphologies in vitro. These cells were treated with recombinant human bone morphogenetic proteins‐2 and ‐4 to determine the differentiation‐inducing activities of bone morphogenetic protein on these cells. The mesenchymal stem cells were cultured in medium with 10% preselected horse serum containing 0 to 100 ng/ml recombinant human bone morphogenetic proteins‐2 or ‐4 for a maximum of 4 weeks. Control cultures maintained the stellate morphology of mesenchymal stem cells. Cultures treated with recombinant human bone morphogenetic protein‐2 exhibited discrete cartilage nodules and mineralized bone nodules. The first increase in chondrogenesis was seen at 0.5 ng/ml. Cultures treated with recombinant human bone morphogenetic protein‐4 also exhibited an increase in chondrogenesis at the higher concentration of 2 ng/ml. Skeletal myotubes and adipocytes also appeared in cultures treated with either bone morphogenetic protein. Mesenchymal stem cells do respond to inductive factors, but bone morphogenetic proteins‐2 and ‐4 were not specific for the induction of cartilage and bone.

The Effect of Vitamin a Deficiency and Fluoride on Glycosaminoglycan Metabolism in Bone

The effects of fluoride intake and vitamin A deficiency on glycosaminoglycan metabolism in vivo were investigated. Weanling female rats were fed either a vitamin A deficient diet ad libitum, a vitamin A supplemented diet pair-fed to the deficient animals, or the vitamin A supplemented diet ad libitum. Additionally, each vitamin A dietary group was divided into three subgroups with the animals receiving water containing 0, 10 or 50 ppm fluoride. The results showed that the groups receiving 10 and 50 ppm fluoride at all dietary levels of vitamin A had significantly higher in vivo 35SO4 incorporation in both the epiphyseal and diaphyseal regions of the bone than the animals receiving 0 ppm fluoride. The vitamin A deficient animals incorporated significantly less 35SO4 into glycosaminoglycans in the epiphyseal and diaphyseal regions of the bone compared to the pair-fed, vitamin A sufficient animals for all three fluoride receiving groups. There was no interaction between fluoride intake and dietary vitamin A levels on 35SO4 incorporation into glycosaminoglycans. Fluoride either increased sulfation or turnover of glycosaminoglycans.

A novel method for the serum-free plating of stage-24 chick limb bud cells

A modification of the standard plating of Stage 24 chick limb bud cells in serum-containing media (SCM) is described in which the cells are incubated in SCM for 3 hours before direct plating in a serum-free chemically defined media (SFM). This results in equal plating efficiencies to those obtained with plating in SCM and may allow for more defined culture conditions when testing mitogenic growth factors found in serum.