1. Circulating Osteogenic Cells in Periarticular Non-hereditary Heterotopic Ossification Robert J. Pignolo, M.D., Ph.D. University of Pennsylvania

2. Dysfunctional bone formation: Errors in time and place Age Location Soft Tissue Skeleton YoungOld Normal Bone Formation & Remodeling Heterotopic Ossification Heterotopic Ossification Osteoporosis

3. Clinical Settings for Non-Hereditary Forms of Heterotopic Ossification Pignolo & Foley CRBMM 3: 261-6 (2005)

4. Conditions that Predispose to Non- Heredity Heterotopic Ossification Post-arthroplasty Traumatic brain injury Spinal cord injury CVA Non-neurologic trauma Pignolo & Foley CRBMM 3: 261-6 (2005)

5. Periarticular involvement of non-hereditary HO after traumatic brain injury Garland, DE et al JBJS Am 62: 1143-46 (1980)

6. Circulating Osteogenic Precursor (COP) Cells Circulating mononuclear cells Plastic/glass adherence Markers of osteoblast- like cells Hematopoietic origin Ability to form a mineralized matrix in vitro or bone in vivo. Found in early lesions of heterotopic ossification in FOP Suda et al Stem Cells 27: 2209-19 (2009)

7. COP cells are present in pre-osseous fibroproliferative lesions of FOP CD45Type I ColCD45 + Type I Col + DAPI Suda et al Stem Cells 27: 2209-19 (2009)

8. Col ICD 45Merge +DAPI Standard Confocal Pre-immune COP cells are present in pre-osseous fibroproliferative lesions of FOP Suda et al Stem Cells 27: 2209-19 (2009)

9. COP cells are present in lesions of NHHO Trauma CVA THA TBI SCI Pre- immune Merge + DAPI Col 1CD45

10. COL 1DAPI + Merge Brain Lymph Node COP cells are absent in unaffected tissue

11. Stages of NHHO lesion formation Early stages Late stages Mature bone Infiltration into Soft tissue Cartilage formation Fibroproliferation Neovascularity Perivascular Lymphocytic Infiltration

12. 400X CollagenCD 45 COP cells are found in early stage lesions Merge with DAPI

13. 200X400X CD45 OCN DAPI + Merge CD45 + OCN + COP Cells

14. COP cells near blood vessel CD45/OCN near blood vessel - trauma CD45…Red OCN….Green DAPI.…Blue

15. COP Cells in Peri-articular Non- hereditary HO Lesions DiagnosisAgeSexCOP cells per HPF (%) Stage of Lesion TBI36M2Fibroproliferative 40M7Fibroproliferative 53M6Fibroproliferative SCI25M4Fibroproliferative 42M8Neovascular Trauma21M6Fibroproliferative 30M3Fibroproliferative 38M14Fibroproliferative 53M2Fibroproliferative Arthroplasty52M5Neovascular 80F6Fibroproliferative 84F2Fibroproliferative CVA44F7Neovascular

16. COP cell involvement in HO Endochondral bone formation Heterotopic Bone INJURY INFLAMATION Soft Tissue (non-skeletal) COP cells Release of inflammatory cytokines, migratory factors (e.g., SDF-1) Bone Marrow CXCR4 Pignolo & Foley CRBMM 3: 261-6 (2005) Kan L et al Stem Cells 27: 150-6 (2009) Otsuru S et al BBRC 354: 453-8 (2007) Otsuru S et al Stem Cells 26: 223-34 (2008) Suda RK et al Stem Cells 27: 2209-19 (2009)

17. Summary COP cells are present in lesions of periarticular non-hereditary heterotopic ossification (NHHO) COP cells are associated with early fibroproliferative and neovascular stages of lesion formation in HO, but absent in regions of unaffected tissue (no HO) Osteogenic cells in the blood home to distant sites where they are associated with formation of HO caused by diverse traumatic conditions.

18. Acknowledgements Laboratory: Kevin Egan Elizabeth Russell Lakshman Singh Robin Suda Jung-Hoon Kim Haitao Wang Jill Brodsky Collaborators: Brad Johnson, Univ of Penn Yongwon Choi, Univ of Penn Eric Brown, Univ of Penn Alex Wright, Univ of Penn Steve Emerson, Haverford Univ Peter Adams, Beatson Institute Mary Ann Keenan, Univ of Penn John Esterhai, Univ of Penn Samir Mehta, Univ of Penn Funding: