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).


METHODS
Human dermal fibroblasts (hDFs) were cultured in porous, three-dimensional (3D) collagen sponges with or without chondroinductive DBP. In some experiments, lithium chloride (LiCl), an agonist of the Wnt/beta-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.
Yates
Karen E
KE
Department of Orthopedic Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA. kyates@partners.org
D018775:Bone Demineralization Technique; D001842:Bone and Bones / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D020219:Chondrogenesis / Q000187:drug effects / Q000502:physiology*; D003094:Collagen; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D011208:Powders; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014020:Tissue Extracts / Q000494:pharmacology; D051153:Wnt Proteins
D011208:Powders; D011518:Proto-Oncogene Proteins; D014020:Tissue Extracts; D051153:Wnt Proteins; D003094:Collagen