title: Co-culture of osteoblasts with immature dural cells causes an increased rate and degree of osteoblast differentiation.
abstract: For decades surgeons have exploited the ability of infants to reossify large calvarial defects. To demonstrate the role of dura mater-osteoblast communication during the process of calvarial reossification, the authors used a novel in vitro system that recapitulates the in vivo anatomic relationship of these cell populations. Primary cultures of osteoblast cells from 2-day-old Sprague-Dawley rat pups were grown on six-well plates, and cultures of immature, non-suture-associated dura mater cells from 6-day-old Sprague-Dawley rat pups were grown on Transwell inserts. When the osteoblast and dura mater cell cultures reached confluence, they were combined. This Transwell co-culture system permitted the two cell populations to grow together in the same well, but it prevented direct cell-to-cell contact. Therefore, the authors were able to determine, for the first time, whether paracrine signaling from immature, non-suture-associated dura mater could influence the biologic activity of osteoblasts. Osteoblasts co-cultured with dural cells proliferated significantly faster after 2 days (2.1 x 10(5) +/- 2.4 x 10(4) versus 1.4 x 10(5) +/- 2.2 x 10(4), p < or = 0.05) and 4 days (3.1 x 10(5) +/- 5 x 10(4) versus 2.2 x 10(5) +/- 4.0 x 10(4), p < or = 0.01) than did osteoblasts cultured alone. After 20 days, co-cultured osteoblasts expressed greater amounts of mRNA for several markers of osteoblast differentiation, including collagen I alpha I (4-fold), alkaline phosphatase (2.5-fold), osteopontin (3-fold), and osteocalcin (4-fold), than did osteoblasts cultured alone. After 30 days, co-cultured osteoblasts produced bone nodules that were significantly greater both in number (324 +/- 29 nodules versus 252 +/- 29 nodules per well, p , < or = 0.04) and total area of nodules (65 +/- 11 mm(2) versus 24 +/- 1.6 mm(2), p < or = 0.003) than osteoblasts cultured alone. To begin to understand how dural cells effect changes in osteoblast gene expression, the authors compared the expression of candidate genes, transforming growth factor beta 1 and fibroblast growth factor 2, in dural cells and osteoblasts before and after 5 days of culture. Interestingly, the dura mater produced marked amounts of these osteogenic cytokines compared with osteoblasts.The described co-culture system demonstrated that co-cultured osteoblasts proliferated more rapidly and experienced an increased rate and degree of cellular maturation than did osteoblasts cultured alone. The authors hypothesize that this effect was due to paracrine signaling (e.g., transforming growth factor beta 1 and fibroblast growth factor 2) from the dura mater, and they are investigating those mechanisms in ongoing experiments. Collectively these data verify that immature, non-suture-associated dura mater can influence the biologic activity of osteoblasts. Moreover, the production of cytokines derived from the dura mater (e.g., transforming growth factor beta 1 and fibroblast growth factor 2), and they may begin to explain why immature animals and infants with intact dura mater can reossify large calvarial defects.
authors.affiliation: Department of Surgery, Stanford University School of Medicine, CA 94305-5148, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Spector
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Greenwald
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Warren
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Bouletreau
authors.forename: Francesca E
authors.initials: FE
authors.lastname: Crisera
authors.forename: Babak J
authors.initials: BJ
authors.lastname: Mehrara
authors.forename: Michael T
authors.initials: MT
authors.lastname: Longaker
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001861:Bone Regeneration; D002454:Cell Differentiation; D002455:Cell Division; D018920:Coculture Techniques; D024042:Collagen Type I / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004388:Dura Mater / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D053495:Osteopontin; D019899:Paracrine Communication; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012795:Sialoglycoproteins / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1
chemical_list: D024042:Collagen Type I; D016207:Cytokines; D012333:RNA, Messenger; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; D015675:Osteocalcin; D053495:Osteopontin; D000469:Alkaline Phosphatase