1. Osteonecrosis of the Femoral Head Valérie Gangji Rheumatology and Physical Medicine Hôpital Erasme Université Libre de Bruxelles Belgium 1

2. Cellular Therapy for osteonecrosis Osteonecrosis of femoral head – Painful disorder leading in its late stage to fracture and total hip replacement – Corticosteroids and alcohol abuse are among the most widely recognized risk factor in Caucasians – Sickle cell disease is the major risk factor for ON in African patients – The prevalence of ON in sickle cell patients – For symptomatic ON: 3-10% – For asymptomatic ON : 10-40%

3. Cellular therapy for osteonecrosis Osteonecrosis of the femoral head ON is more frequent in homozygous patient (hemoglobin SS) but ON can also be found in heterozygous patients and in thalassemia α et β The risk of ON is correlated to the incidence of vaso-occlusive crisis and to high hematocrit Painful disorder leading in its late stage to fracture and total hip replacement Core decompression of the femoral head is the most widespread procedure to treat early stages of ON Efficacy of core decompression remains controversial

4. Cellular therapy for osteonecrosis

6. Ostéonécrose de la tête fémorale Physiopathogénie : Hypothèse vasculaire Traumatisme Coagulation Embolie Hyperpression intravasculaireintramédullaire Interruption Thrombose Compression vasculaireextravasculaire Diminution du flux sanguin Ischémie OSTEONECROSE

7. Osteonecrosis - physiopathology Cellular therapy Vascular and Bone Disease Vascular Disease Bone disease Fat emboli Into capillaries Intravascular coagulation Osteoblastic Cells Therapeutic Strategies Cellular Therapy Therapeutic Strategies Core Decompression 7 Stems cells

8. Osteonecrosis in sickle cell anemia Cellular therapy Autologous bone marrow transplantation was reported for the first time in 1994 in a patient sustaining ON of the humeral head due to SS anemia Three months after the transplantation, MRI showed a tendancy towards normalisatio of the signal.

9. Osteonecrosis - cellular based therapy Cartilage-tendon 9

10. Concentration Expansion Expansion and differentiation Bone marrow is concentrated for immediate implantation Bone marrow stem cells are separated and expanded for 1 to 3 weeks Bone marrow stem cells are separated, expanded and differentiated in a specific cell population 10 Osteonecrosis-cellular based therapy

11. Cellular therapy for osteonecrosis Bone marrow procedure Bone marrow aspiration from the posterior iliac crest Cobe system - bone marrow collection kit Harvest system - bone marrow collection kit

12. Cellular therapy for osteonecrosis Bone marrow procedure Bone marrow is filtered to eliminate spicules and bacteria

13. Cellular therapy for osteonecrosis Bone marrow implantation Gangji et al. 2005, Marker et al. 2008, Mont et al. 2004, Song et al. 2007 13 Less invasive surgery, small incision, 3mm core decompression, injection of the concentrated bone marrow through the trephine-lower morbidity and postoperative complication rates

14. Cellular therapy for osteonecrosis A five year prospective controlled double blind trial on the efficacy of bone marrow implantation in osteonecrosis of the femoral head 19 patients suffering from stage 1 and 2 ON of the femoral head Patient’s hips (24 hips) were alternatively allocated to a core decompression procedure only (control group) or with autologous bone marrow grafting (bone marrow graft group) Primary outcomes were safety clinical symptoms disease progression from stage 1-2 to the stage 3 14

15. Cellular therapy for osteonecrosis Assessment at baseline, 3, 6, 12, 24, 36, 48 and 60 months Clinical evaluation visual analogue scale (VAS), algofunctionnal index of Lequesne, WOMAC score Radiological evaluation anteroposterior radiographs of the affected hip measurement of the necrotic zone by MRI on T1-weighted scans

16. Cellular therapy for osteonecrosis Bone marrow harvest procedure 400 ml of bone marrow obtained from the anterior or posterior iliac crest Mononuclear cells sorted on a Cobe Spectra cell separator and marrow concentrated to a final volume of ~ 50 ml Total injected volume was 51  1.8 ml Number of leukocytes 2.0 10 9  0.3 10 9 Number of CD34+ cells 1  0.2 % Number of CFU-F 92  28 / 10 7 cells

17. Cellular therapy for osteonecrosis Core decompression Bone marrow implantation

18. Cellular therapy for osteonecrosis Homing of bone marrow cells at 24H Leukocytes were labeled with Indium Oxine and mixed with the injected bone marrow

19. Cellular therapy for osteonecrosis Efficacy on symptoms * *** * Gangji et al. 2004, Gangji et al. 2009 - Submitted

20. Cellular therapy for osteonecrosis Efficacy on symptoms * * ** * Gangji et al. 2004, Gangji et al. 2009 - Submitted

21. Cellular therapy for osteonecrosis Efficacy on symptoms Gangji et al. 2004, Gangji et al. 2009 - Submitted

22. Cellular based therapy for osteonecrosis Efficacy on disease evolution Time to Collapse - Kaplan-Meier Survivorship Analysis Gangji et al. 2004, Gangji et al. 2009 - Submitted 3 of 13 hips in the bone marrow graft group progressed to stage III. Log-rank test; p=0.008 7 of 11 hips in the control group deteriorated to the stage III 22

23. Cellular based therapy for osteonecrosis Efficacy on volume of the lesion * The volume of the necrotic lesion decreased significantly in the bone marrow graft group at 24 months (p=0.041) and approached statistical significance at 60 months (p=0.066) Gangji et al. 2004, Gangji et al. 2009 - Submitted

24. Cellular based therapy for osteonecrosis Results of previous studies Hernigou et al. (2002) – Prospective study: 189 hips in 116 patients – Followed up from 5-10 years – Total hip replacement was needed : in 5/145 hips for stage 1-2 25/44 hips for stage 3-4 – More efficient in SS patients – Efficacy related to the amount of CFU implanted

25. Cellular based therapy for osteonecrosis Osteonecrosis – Hypothesis for efficacy Vascular and Bone Disease Vascular Disease Osteonecrosis Fat emboli Into capillaries Intravascular coagulation Mesenchymal Stem Cells Osteoblastic Cells Therapeutic Strategies Bone marrow Therapeutic Strategies Core decompression 25

26. Cellular therapy for bone diseases Hypothesis for efficacy Availability of mesenchymal and endothelial stem cells endowed with osteogenic and angiogenic properties could explain the efficacy The efficacy of BMPs like BMP-2 and BMP-7 in treating nonunion fracture could be explained by their ability to recruit mesenchymal stem cells and to initiate their differentiation into osteoprogenitors Bone marrow will also provide osteogenic and angiogenic growth factors like FGF-2, TGF-β, PDGF, VEGF, angiopoietin resulting in increased osteogenesis and angiogenesis

27. Cellular therapy for osteonecrosis Conclusions Pioneer trials in bone marrow implantation in osteonecrosis have shown safety and some degree of efficacy Larger controlled and randomized trials are needed to confirm those results Improvement of clinical outcome can only be possible through the optimization of the cellular product – Improvement of intra-operative bone marrow harvest/concentration – Selection of cells

28. Remerciements L’équipe de l’unité de thérapie cellulaire de l’Hôpital Erasme

29. Cellular therapy for osteonecrosis Conclusions Improvement of clinical outcome can only be possible through the optimization of the cellular product – Improvement of intra-operative bone marrow harvest/concentration – Selection of cells PREOB® - BONE THERAPEUTICS