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Demineralized Bone Alters Expression of Wnt Network Components During Chondroinduction of Post-Natal fibroblasts Karen E

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Demineralized Bone Alters Expression of Wnt Network Components During Chondroinduction of Post-Natal fibroblasts Karen E OsteoArthritis and Cartilage (2004) 12, 497–505 © 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2004.02.009 International Cartilage Repair Society Demineralized bone alters expression of Wnt network components during chondroinduction of post-natal fibroblasts Karen E. Yates Ph.D* Department of Orthopedic Surgery, Brigham and Women’s Hospital, and Skeletal Biology Research Center, Massachusetts General Hospital, Boston, USA Summary 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 chondroinduc- tive DBP. In some experiments, lithium chloride (LiCl), an agonist of the Wnt/-catenin signaling pathway, was added to the culture media. Sponges were cultured for intervals (0.5–21 days) before processing for molecular, histologic, and biochemical analyses. Expression of wnt, fzd, and sfrp genes was characterized by semi-quantitative RT-PCR. Fibroblasts’ contacts with DBP were documented by histology. Accumulation of proteoglycan in extracellular matrix was evaluated by histology (metachromasia in toluidine blue-stained sections) and quantitative immunoassay (chondroitin 4-sulfate ELISA). Results: Expression of 15 wnt, fzd, and sfrp family members was detected in hDFs by RT-PCR. A subset of those genes (wnt2b, wnt5b, wnt10b, fzd6, fzd7) showed altered expression in hDFs exposed to DBP for 3 days. wnt and fzd gene expression was not altered before hDFs contacted the DBP within the collagen sponge. Human DFs cultured in plain collagen sponges and treated with LiCl accumulated significantly more metachromatic matrix than NaCl-treated controls on day 10, and showed a trend towards increased matrix chondroitin-4 sulfate content. Conclusions: These data suggest that changes in Wnt signaling contribute to chondroinduction of post-natal fibroblasts by DBP. This is the first evidence that Wnt components, which are essential regulators of pre-natal chondrocyte differentiation, may also influence post-natal chondrocyte differentiation induced by DBP. © 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Wnt, Demineralized bone, Dermal fibroblast, Chondroinduction. Introduction particles approximates the architecture of DBP in in vivo implants9. With hDFs used as target cells, the DBP/ The wnt gene family encodes secreted proteins that inter- collagen sponge system models an early step in DBP- act with cellular receptors encoded by the fzd gene family. induced endochondral osteogenesis: the phenotypic Together with secreted, fzd-related proteins (encoded by conversion of mesenchymal cells into chondroblasts10,11. sfrp genes) that act as Wnt antagonists, these proteins Seven days after hDFs are seeded onto DBP/collagen form a regulatory network that profoundly influences sponges, cells that have contacted the DBP show accumu- chondrocyte differentiation and chondrogenesis during em- 12 1–8 lation of metachromatic extracellular matrix and gene bryonic skeletal development . Whether Wnt, Fzd, and expression of the cartilage matrix components, collagen Sfrp (hereafter referred to as Wnt components) similarly type II and aggrecan13. Prior to the expression of cartilage influence post-natal chondrocyte differentiation and chon- phenotypic genes, DBP induces shifts in expression of drogenesis, however, is not well understood. several functional classes of genes in hDFs, including In an experimental system of post-natal chondrocyte matrix proteins, cytoskeletal elements, protein synthesis differentiation, human dermal fibroblasts (hDFs) are cul- machinery, signal transduction proteins, and peptide tured with demineralized bone powder (DBP) in a porous growth/differentiation factors14,15. For some genes, DBP- collagen sponge9. Demineralized bone is used clinically induced changes in expression are transient and their because of its ability to induce skeletal tissue by an mRNAs return to control levels by day 7, when the chondro- endochondral mechanism10,11.Inthein vitro culture de- cyte phenotype is evident14. Altered expression of other vice, a portion of DBP is held in place between two thin genes is sustained during accumulation of cartilage-like layers of collagen. This arrangement of demineralized bone matrix (chondrogenesis) on days 7–21. These patterns of altered gene expression begin to define cellular stages *Address correspondence and reprint requests to: Karen E. of chondroblast differentiation and chondrogenesis in the Yates Ph.D., Orthopedic Research, Brigham and Women’s Hospi- 15 tal, 75 Francis Street, Boston MA 02115, USA. Tel: 617 732-6943; collagen sponge culture system . Fax: 617 732-6705; E-mail: [email protected] The importance of Wnt components in pre-natal Received 22 April 2003; revision accepted 24 February 2004. chondrocyte differentiation suggested the hypothesis that 497 498 K. E. Yates: Alteration of Wnt components by demineralized bone this network also regulates post-natal chondrocyte differen- A preliminary experiment was performed to determine an tiation. The objective of this study was to determine appropriate concentration of LiCl for sponge experiments. whether Wnt signaling contributes to chondroinduction by Monolayer cultures of hDFs exposed to 5 mM LiCl or DBP. Therefore, the DBP/collagen sponge system was control NaCl for 10 days were viable. Greater concen- used to characterize Wnt component gene expression trations of LiCl (10 or 20 mM) were toxic. Therefore, 24 h during chondroblast differentiation and chondrogenesis. after sponges were seeded with hDFs, 4 µl of LiCl or NaCl The contribution of one aspect of Wnt signal transduction stock solution (5 M) was added to each well and mixed into to post-natal chondrocyte differentiation was assessed the culture media by pipetting. Fresh media containing by exposing hDFs in collagen sponges to lithium chloride 5 mM LiCl or NaCl was added 2 days later when the (LiCl), an agonist of the Wnt/-catenin signaling sponges were removed from the seeding chambers, and pathway16. every 3–4 days thereafter. Ten days after seeding, the sponges were harvested for histology and proteoglycan extraction. Methods CELLS RNA ISOLATION Human dermal fibroblasts were isolated by outgrowth Total RNA was prepared from monolayer cultures by from skin explants (neonatal foreskins) and cultured in lysing cells in Trizol reagent (Invitrogen, Carlsbad, CA). monolayer to passage #7 before use12. Basal culture Groups of control collagen or DBP/collagen sponges were pooled and homogenized in Trizol reagent (1 ml per media for all hDF experiments consisted of Dulbecco’s 14 MEM, 10% fetal bovine serum, 100 U/ml penicillin, and 100 sponge) . RNA quality was evaluated by absorbance µg/ml streptomycin. Os Te (osteogenesis imperfecta em- readings at 260 and 280 nm, and by electrophoresis on bryonic human dermal fibroblasts, #CRL-1262) and MG63 formaldehyde-agarose gels. cells (human osteosarcoma, #CRL-1427) were purchased and cultured as recommended by the supplier (American CDNA SYNTHESIS Type Culture Collection, Manassas, VA). Total RNA was treated with DNase I (Roche Molecular Biochemicals, USA) to eliminate any contaminating ge- CHONDROINDUCTION SYSTEM nomic DNA. Aliquots of 5 µg of DNase-treated RNA were Demineralized bone powder was prepared from rat long used in random hexamer-primed cDNA synthesis reactions bones, as described17. Three-dimensional DBP/collagen or (100 µl total volume) according to the manufacturer’s in- plain collagen sponges were prepared as described9.In structions (Invitrogen). One µl of cDNA (the equivalent of brief, to prepare bilaminate sponges, 120 µl of 0.5% type I 50 ng total RNA) was used in each PCR. collagen solution (Cellagen PC-5, ICN Biomedicals, Costa Mesa, CA) was neutralized with 0.01 M HEPES (pH 7.4) PCR PRIMERS and 0.01 M NaHCO3, poured into a mold, and frozen. A spacer of moistened paper was placed over the frozen Primers specific for the housekeeping gene collagen layer and a second aliquot of collagen (130 µl) glyceraldehyde-3-phosphage dehydrogenase (G3PDH) was added. The construct was frozen, lyophilized and were purchased (Clontech, Palo Alto, CA). Published irradiated with ultraviolet light (3 h per side). The spacer primers were used for wnt2b, wnt10b, fzd2, fzd518 and paper was removed and a 3-mg portion of DBP was placed wnt16a, wnt16b19 (Table I). Additional primers (Table I) between the layers of bilaminate sponges. To prepare plain were designed using the Primer3 program20 (code avail- collagen sponges, a single layer of collagen (250 µl) was able at http://www-genome.wi.mit.edu/genome_software/ poured into a mold, frozen, lyophilized and irradiated with other/primer3/html). A previous study showed that a ultraviolet light. specific wnt5a mRNA that has a unique 3′ untranslated region (UTR) is induced in hDFs cultured with DBP21. Because 3′ UTRs are incompletely catalogued, PCR COLLAGEN SPONGE SEEDING AND CULTURE CONDITIONS primers were designed to target protein-coding regions. For cell seeding, each sponge was positioned in a Primers were
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