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Co-Culture of Osteoblasts with Immature Dural Cells Causes an Increased Rate and Degree of Osteoblast Differentiation

Spector, Jason A. M.D.; Greenwald, Joshua A. M.D.; Warren, Stephen M. M.D.; Bouletreau, Pierre J. M.D.; Crisera, Francesca E. B.A.; Mehrara, Babak J. M.D.; Longaker, Michael T. M.D.

Plastic and Reconstructive Surgery: February 2002 - Volume 109 - Issue 2 - p 631–642
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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-tocell 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 × 105 ± 2.4 × 104 versus 1.4 × 105 ± 2.2 × 104, p ≤ 0.05) and 4 days (3.1 × 105 ± 5 × 104 versus 2.2 × 105 ± 4.0 × 104, p ≤ 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 IαI (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 ≤ 0.04) and total area of nodules (65 ± 11 mm2 versus 24 ± 1.6 mm2, p ≤ 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 β1 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 cocultured 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 β1 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 β1 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. (Plast. Reconstr. Surg. 109: 631, 2002.)

Stanford, Calif.

Michael T. Longaker, M.D. Department of Surgery Stanford University School of Medicine 269 Campus Drive CCSR, Room 1225-S Stanford, Calif. 94305-5148Longaker@Stanford.edu

From the Department of Surgery, Stanford University School of Medicine. Received for publication January 24, 2001; revised April 19, 2001.

©2002American Society of Plastic Surgeons