Harnessing the Osteogenic Capability of Neonatal Dura to Heal Critical Sized Defects

1. Harnessing the Osteogenic Capability of Neonatal Dura to Heal Critical Sized Defects Walter Sweeney B.S., Brendan Alleyne B.S., Adam Cash M.D., Christy Gliniak B.S., Gregory Cooper PhD., Arun Gosain M.D. Case Western Reserve University, Department of Plastic & Reconstructive Surgery, Cleveland, Ohio Presenter: Brendan Alleyne B.S.

2. Critical Sized Defects Defects incapable of healing in the animals lifetime Problematic for adults and children because vital structures are unprotected Interestingly, children < 1 year of age can heal large cranial defects Healing is due to osteogenic potency of dura but potency decreases with age Can the dural osteogenic potency present in children be harnessed to heal critical sized defects? Introduction

3. Disclosure Nothing to disclose

4. Rat neonatal dura transplanted into adult rat critical sized defects would retain the ability to induce osteogenesis and augment healing. Hypothesis

5. 24 Adult Rats 24 Neonatal Rats Materials and Methods Craniotomy with dural harvest from parietal aspect of animals 8, 17, and 45 days PN 45 days PN (n=6) 8 days PN (n=6) Untreated 17 days PN (n=6) 60 day old Adult rats with 2.3mm defect 60 day old adult rats with 2.3mm defect 60 day old adult rats with 2.3mm defect 60 day old Adult rats with 2.3mm defect

6. Materials and Methods Dura Craniotomy 8,17,or 45 day old postnatal rat Inlayed

7. Micro-CT images performed 5 weeks post-operatively Image J was used to assess pixel density as a surrogate for bone formation Pixel density was compared between the cranial defect and intact cranial bone At 5 weeks only untreated defects and defects treated with 8 day old neonatal dura were evaluated Radiographic images performed 8 weeks post-operatively Image J was used to measure the Bone Gap Bone gap was defined as the distance between bone formation and edge of the defect At 8 weeks the bone gap was measured for all animals Analysis

8. Results • Micro-CT 5 weeks Untreated Treated w/ 8 day old dura

9. Results • Radiograph 8 wks Treated w/ 8 day old dura Untreated

10. Results of Animals Evaluated with Micro-CT at 5 Weeks Increased Pixel Density = Increased Bone Formation • Defects treated with 8 day old dura achieved pixel density equivalent to intact cranial bone • Pixel density of untreated defects was decreased 3 fold as compared to intact cranial bone

11. Results of Animals Evaluated with Radiographs at 8 Weeks Decreased Bone Gap = Increased Bone Formation • Defects treated with 8 day old dura had the smallest bone gap and most bone formation • Bone gap was largest in untreated defects • Supporting evidence that dura becomes less osteogenic with age, the bone formation decreased as the age of the dura increased

12. First study to demonstrate the neonatal dura retains osteogenic potency following transplantation This study supports that the osteogenic capability of dura decreases with age Conclusion

13. Significance of the findings… • Dura can be harnessed to augment CSD healing • Future studies will focus on identifying growth factors responsible for cranial regeneration • Such findings will facilitate the engineering of biocompatible grafts capable of mimicking dural induced osteogenesis • Additionally, current studies aim to determine if fetal dura has an even greater degree of osteogenic potential than that of perinatal dura

14. Dr. Gosain Walter Sweeney Christy Gliniak Davood Varghai Sincere thanks to…

15. Harnessing the Osteogenic Capability of Neonatal Dura to Heal Critical Sized Defects Walter Sweeney B.S., Brendan Alleyne B.S., Davood Varghai M.D., Adam Cash M.D., Christy Gliniak B.S., Gregory Cooper PhD., Arun Gosain M.D.Case Western Reserve University, Department of Plastic & Reconstructive Surgery, Cleveland, Ohio Results Conclusions Introduction • itical Sized Defects • Critical Sized Defects are defects incapable of healing in the animals lifetime • These are problematic for adults and children because vital structures are left unprotected • Interestingly, children < 1 year of age retain the ability to heal large cranial defects • Healing is due to the osteogenic potency of dura, however, potency decreases with age • Can the dural osteogenic potency present in children be harnessed to heal critical sized defects? • While the osteogenic properties of dura are thoroughly documented, the ability of dura to augment critical sized cranial defect (CSD) healing is less established • In the present study we evaluated whether transplanted dura retained sufficient osteogenic potential to aid in CSD repair This is the first study to demonstrate that neonatal dura retains osteogenic potency following transplantation. This study supports that the osteogenic capability of dura decreases as the animal ages. • Increased Pixel Density : Increased Bone Formation • Defects treated with 8 day old dura achieved pixel density equivalent to intact cranial bone • Pixel density of untreated defects was decreased 3 fold as compared to intact cranial bone Methods Future Investigation This study suggests that the in vivo osteogenic properties of dura are preserved with transplantation and can be harnessed to augment CSD healing. Future studies will focus on identifying factors responsible for cranial regeneration and engineering biocompatible grafts capable of mimicking dural induced osteogenesis. Additionally, future studies will aim at seeing if fetal dura has an even greater degree of osteogenic potential. Craniotomies were performed and 5x5 mm of parietal dura harvested from twenty-four Sprague Dawley rats at 8, 17, and 45 days postnatal. A 2.3 mm CSD was created in the parietal bone of twenty-four 60 day old Sprague Dawley rats. Animals were randomized into four experimental groups: A. 8-day old dura inlayed into CSD; B. 17-day old dura inlayed into CSD; C. 45 day old dura inlayed into CSD; or D. Untreated CSD. Four animals treated with 8-day old dura were evaluated five weeks post-operatively using micro-CT and the remaining animals were evaluated 8 weeks post-operatively with radiographs. Micro-CT images were analyzed for pixel density within the CSD. Radiographic images were evaluated by measuring the distance between new bone formation within the CSD and the edge of the cranial defect; we defined this as the bone gap. • Decreased Bone Gap : Increased Bone Formation • Defects treated with 8 day old dura had the smallest bone gap and most bone formation • Bone gap was largest in untreated defects • Supporting evidence that dura becomes less osteogenic with age, the bone formation decreased as the age of the dura increased Analysis References • Moreira-Gonzalez, A., Jackson, I. T., Miyawaki, T., Barakat, K. & DiNick, V. Clinical outcome in cranioplasty: • critical review in long-term follow-up. J. Craniofac. Surg. 14, 144-153 (2003). • Chim, H. & Gosain, A. K. Biomaterials in craniofacial surgery: experimental studies and clinical application. J. Craniofac. Surg. 20, 29-33 (2009). • Cho, Y. R. & Gosain, A. K. Biomaterials in craniofacial reconstruction. Clin. Plast. Surg. 31, 377-85, v (2004). • David, L., Argenta, L. & Fisher, D. Hydroxyapatite cement in pediatric craniofacial reconstruction. J. Craniofac. Surg. 16, 129-133 (2005). • David, D. J. & Cooter, R. D. Craniofacial infection in 10 years of transcranial surgery. Plast. Reconstr. Surg. 80, 213-225 (1987). • Fearon, J. A. et al. Infections in craniofacial surgery: a combined report of 567 procedures from two centers. Plast. Reconstr. Surg. 100, 862-868 (1997). • Hobar, P. C., Masson, J. A., Wilson, R. & Zerwekh, J. The importance of the dura in craniofacial surgery. Plast. Reconstr. Surg. 98, 217-225 (1996). • Li, S., Quarto, N. & Longaker, M. T. Dura mater-derived FGF-2 mediates mitogenic signaling in calvarialosteoblasts. Am. J. Physiol. Cell. Physiol. 293, C1834-42 (2007). • Gosain, A. K. et al. Quantitative assessment of cranial defect healing and correlation with the expression of TGF-beta. J. Craniofac. Surg. 12, 401-404 (2001). (1) 8,17,or 45 day old postnatal rat (2) Isolated calvaria Untreated Treated w/ 8 day old dura (3) Isolated dura mater (4) Inlayed dura mater Untreated Treated w/ 8 day old dura