Introduction 10/12/2015 DISCLOSURE. Nucleus/Disc Repair Techniques: (I) Cellular therapy (II) Growth factor therapy (III) Gene therapy 11/21/15

Transcription

1 10/12/2015 Biological Disc Regeneration and Repair in the Spine Physiology of Disc Degeneration and Biologic Regeneration and Repair Options Dom Coric, M.D. Carolina Neurosurgery and Spine Associates Chief, Department of Neurosurgery Carolinas Medical Center Charlotte, NC 11/21/15 DISCLOSURE Medtronic: Consultant Spine Wave: Consultant/Stock/Royalties Globus Medical: Consultant Premia Spine: Consultant DiscGenics: Consultant All disc/nucleus repair procedures are investigational. Introduction Nucleus/Disc Repair Techniques: (I) Cellular therapy (II) Growth factor therapy (III) Gene therapy Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Pfirrmann CS, Boos N, et al: Magnetic resonance imaging classification of lumbar intervertebral disc degeneration. Spine 26: ,2001 1

2 10/12/2015 Annulus Fibrosus Outer fibrous structure with: (A) Fibroblast-type cells Scant cells (B) Type I collagen (abundant) Tensile strength. IVD Fibro-cartilaginous structure. Nucleus Pulposus Inner gelatinous structure (A) Chondrocytic cells Proteogylcans (B) Type II collagen Compressive forces. IVD NUCLEUS PULPOSUS 2 cells types derived from distinct embryonic sources (maintain ECM homeostasis): (1) Notochord cells notochordal remnant generally disappear by age 20 (2) Chondrocytic disc cells derived from axial mesoderm Homeostasis: balance between anabolism and catabolism of disc cells and the ECM they produce. ECM Extracellular matrix primarily consists of: (a) Proteoglycans Aggrecan and versican: largest/most common Hydrophilic molecules: protein stems surrounded by highly neg-charged glycosaminoglycan (GAG) side chains. Chondroitin-6-sulfate and keratin sulfate :» 2 most abundant GAG molecules, attract and Aggrecan hold pos-charged H2O molecules. (b) Type II collagen: scaffold 2

3 10/12/2015 IVD Degenerative Disc Disease Loss of chondrocytic nucleus pulposus cells results in inability to produce and maintain normal ECM. Annulus fibrosus Delamination: annular tears. Nucleus pulposus Cellular loss: depletion of extracellular matrix, replacement with fibrocartilage. Dessication: progressive loss of proteoglycans/h2o. IVD DEGENERATIVE DISC DISEASE (DDD) Progressive changes in disc composition and function out of proportion those associated with normal aging. Calcification of cartilaginous endplate (sclerosis) limits blood/nutrient supply. Factors favor ECM destruction (catabolism) over production (anabolism). Balance Imbalance Anabolic Catabolic IVD DEGENERATIVE DISC DISEASE (DDD) Proteoglycans Hydration Disc dessication/degeneration/ disc ht Load on surrounding structures Annular tears/disc dessication, loss of ht/modic changes/hnp PAIN Photos courtesy of Prof Rauschning MD 3

4 10/12/2015 Disc Repair 3 main mechanisms: I. Growth factors: exogenous protein injection. Boost native chondrocytic cell production by upregulating production of anabolic ECM proteins, down-regulate catabolic factors. II. Gene therapy: transfer of genetic material. Boost native chondrocytic cell production by inserting genetic material to maintain/restore ECM. III. Cell therapy: exogenous injection of cells. Introduction of exogenous cells to augment/replenish ECM. Stem, native disc and chondrocyte cells Growth Factors (I) Direct protein (growth factor) injection. Growth factors: small peptide cytokines with cell regulatory function. In vitro studies show exogenous application of growth factors can positively influence ECM synthesis by chondrocytic cells. Growth Factors Up-regulate ECM proteins: (a) Transforming growth factor (TGF-beta) (b) Insulin-like growth factor 1 (IGF-1) (c) Epidermal growth factor (EGF) (d) Platelet-derived growth factor (e) Bone morphogenetic proteins (BMP) BMP-7 (OP-1), BMP-2, GDF-5 Increase anabolic activity. 4

5 10/12/2015 Growth Factors Down-regulate inflammatory cytokines: (a) Interleukin (IL-1, IL-6) (b) Tumor necrosis factor-alpha (TNF) (c) Matrix metalloproteinases (MMPs) (d) Nitric oxide (NO) (e) Prostaglandin E2 (PGE2) Decrease catabolic activity. Growth Factors Challenges Practical clinical use of growth factors for nuclear repair may be limited by their short biologic half-lives (?hours/days). May be especially limited in chronic conditions, such as DDD. Clinical correlation: growth factors clearly improve disc structure in in vivo animal studies, will this correlate with pain improvement in humans? Gene Therapy (II) Transfer of genetic material (DNA or RNA) into a target cell (i.e. chondrocytic cells) to modulate cellular activity (i.e. upregulate anabolic proteins or down-regulate catabolic proteins), potentially long-term. 5

6 10/12/2015 Gene Therapy Challenges Since gene therapy involves active transfer of genetic material, generally utilizing a viral vector, there is some inherent risk (i.e. viral mutagenicity, systemic viral infection, immune response). Therefore, may be more appropriate for potentially lifethreatening disorders, especially those resulting from single gene defects (i.e. cystic fibrosis, sickle cell dz). May play a more limited role in non-life threatening disorders with multifactorial etiology (i.e. rheumatoid arthritis, DDD). Cell Therapy Notochordal cells Allogeneic: embryonic human NP, soon after birth these cells diminish rapidly. Chondrocytes Autologous: mature Allogeneic: juvenile Mesenchymal stem cells Autologous: bone marrow/adipose Allogeneic: embryonic/adult/umbilical Nucleus Repair Cell therapy: Advantages Nucleus is surrounded and contained by annulus, preventing cell migration. Limited blood supply and contained space provides immune privileged milieu. 6

7 10/12/2015 Nucleus Repair Cell therapy: Challenges Degenerative environment: if endplate sclerosis limits blood supply: decrease O2, increase lactic acid (decrease ph) leads to cell death and decreased ECM production, then transplanted cells are subjected to same harsh environment. Normal ECM production and turnover is slow (generally measured in yrs), transplanted cells may take months/yrs to affect change. Nucleus Repair Cell therapy: Mechanism (1) Cell harvest (2) Cell expansion Musculoskeletal cell therapies generally introduce 5-10 million cells/defect: cells are expanded by growing in monolayer to encourage proliferation. (3) Add scaffold/carrier Hyaluronic acid, fibrin, silk, collagen (4) Insertion Ideally minimally invasive with percutaneous needle Cell Therapy Carrier Cell expansion Donor Cells 1 7

8 10/12/2015 CNSA Disc Repair IND Experience NuQu Phase I, Phase II Juvenile chondrocyte nucleus repair Thrombin/fibrinogen carrier Phase I: prospective Phase II: prospective, randomized, placebo Mesoblast Phase II, Phase III (ongoing) Stem cell nucleus repair Phase II/III: prospective, randomized, placebo Allogeneic mesenchymal stem cells Hyaluronic acid carrier Coric D, Pettine K, Sumich A, Boltes MO: Prospective Study of Disc Repair with NuQu Allogeneic Chondrocytes. J Neurosurg-Spine 18:36-42, 2013 Stem Cells for DDD Yoshikawa et al. (2010) reported on two patients treated with expanded iliac crest derived mesenchymal stem cells Case report: 2 patients Autologous marrow-derived mesenchymal stem cells. 100,000 cells/ml Advanced DDD (stenosis and adjacent to fusion). Positive clinical results. Yoshikawa T, Ueda Y, Miyazaki K, Koizumi M, Takakura Y: Disc regeneration therapy using marrow mesenchymal cell transplantation: a report of two cases. Spine 35:E475-80, 2010 Stem Cells for DDD Orozco et al. (2011) published a pilot series of ten patients with chronic LBP also treated with expanded iliac crest derived mesenchymal stem cells. Case series: 10 patients Autologous marrow-derived mesenchymal stem cells. 5,000,000 cells/ml DDD with chronic low back pain (minimum 6 months). Positive clinical results. Orozco L, Soler R, Morera C, Alberca M, Sanchez A, Garcia-Sancho J:Intervertebral disc repair by autologous mesenchymal bone marrow cells: a pilot study. Transplant 92: ,

9 10/12/2015 Cartilage Cells for DDD NuQu: Juvenile cartilage cells Phase I: 15 pts - prospective, non-randomized. Phase II: 44 pts - prospective, randomized, blinded, placebocontrolled. Pts with discogenic back pain secondary to mild/moderate degenerative disc disease (DDD) L2-S1. Fibrin glue carrier. Coric D, Pettine K, Sumich A, Boltes MO: Prospective Study of Disc Repair with NuQu Allogeneic Chondrocytes. J Neurosurg-Spine 18:36-42, 2013 Cartilage Cells for DDD Phase I Pilot Study Levels: L3-4: 2 L4-5: 1 L5-S1: 12 Injection duration: Avg=11.6 s (Range 5-32s) Injection amount: Avg=1.4cc (Range 1-1.6cc) Est # of viable cells million cells/cc Indiscal press (peak): Avg=92.4 psi (Range ) Cartilage Cells for DDD Phase I results: CLINICAL Mean preoperative pain (NRS), disability (ODI) and function (SF-36) scores improved significantly at six months and were maintained through 2 years. Pre-op 6 mths 2 yrs NRS: (p=0.0036) ODI: (p<0.0001) SF-36: (p=0.0014) 9

10 10/12/2015 Disc Repair for DDD CH: Pt is 40 yo with long h/o mechanical LBP. 7 in, 22-gauge. 1.4 cc injection, 12s. Max press= 82 psi. Pt now 1 yr postop resolution of chronic mechanical LBP (16 mnths), no narcotics, VAS=1. Conclusion Disc repair is both a minimally invasive as well as motion preserving technique to treat symptomatic degenerative disc disease earlier and less invasively. Continued investigation into the diagnosis and treatment of DDD is warranted. THANK YOU! 10

11 10/21/2015 Current Trials and the Status of Tissue-Engineering Strategies Roger Härtl, MD Professor of Neurosurgery Director of Spinal Surgery Department of Neurosurgery Weill-Cornell Medical College New York, NY USA Disclosure Consultant Synthes Baxter BrainLab Funding AOSpine AOFoundation NFL Baxter Nuvasive Biological Strategies for Disc Repair and Regeneration Growth Factors Gene therapy Transplantation of cells Stem cells / Progenitor cells Autologous / allogeneic Chondrocytes Autologous / allogeneic juvenile Whole disc allograft transplant Transplantation of disc/scaffold constructs NP Composite bio-engineered discs High density collagen gel 1

12 10/21/2015 DDD and Biological NP Repair Healthy Disc VB Degenerated Disc and Therapeutic Targets VB AF NP AF NP EP Supply Demand EP Metabolism Cells Structure NP AF bone EP Blood Vessel Biomolecules Cells Tissue Engineered Constructs Tissue Engineering for Disc Regeneration Biopsy Growth Factors Genes Mechanical Stimuli Allograft transplant Pluripotent Stem Cells Tissue engineered disc Transplant Progenitor Cells Cell isolation NP AF AC Differentiated Disc Chondrocytes Tissue development Biomaterial implantation Scaffold Cell transplant Cell cultivation Cell proliferation Stages of Disc Repair / Regeneration Degrees of Degeneration Early Intermediate Advanced Viable Cells Structural Damage Therapeutic Strategy Biomolecular Cell-based therapy Engineered constructs 2

13 10/21/2015 Cell implantation Promising Animal Data: MSC Chondrocytes NP cells, etc. Autologous Chondrocytes Euro spine J, , 2008 Canine and clinical trail Autologous cell from AF and NP In canine collected in open procedure For clinical trial collected during discectomy for disc herniation Cells expanded in culture, replanted after 12 weeks Clinical trail 24 month follow-up -112 patients enrolled, patients were randomized - Cells were transplanted 12 weeks after discectomy - OLBPD, VAS, SF-36 scores for follow up - Follow up MRI Eur Spine J 2006,

14 10/21/2015 First (and last) interim analysis Removed cells could be expanded in culture Patients who received cells had greater pain reduction after discectomy at 2 years Discs with transplant demonstrated significant more fluid content on MRI Before 1d 3m 12m 24m 60m Stem Cells 2006 Unexpanded hematopoietic stem cells obtained from iliac crest bone marrow 10 patients 12 months No control No improvement of low back pain at 1 year 8 patients had surgery: Fusion, arthroplasty SPINE Volume 35, Number Two cases - 1. case: adjacent segment disease after ALIF L4/L5 LBP, numbness right lower leg 6 years after fusion - 2. case: Instability with spinal canal stenosis L4/L5 LBP, leg pain over several years 4

15 10/21/2015 MRI outcome 2 Years PRE-OP POST-OP 2yrs PRE-OP POST-OP 2yrs 10 patients with DDD No respond to cons. Therapy after 6months BMA iliac crest, expanded, 12 month outcome VAS and ODI Patients showed improvement Follow up MRI was obtained - No increase of disc height - Significant increase in signal intensity after 1 year month outcome 5

16 10/21/2015 Allogeneic Mesenchymal Progenitor Cells Phase 2 clinical trial 100 patients with moderate to severe low back pain, > 6 months, caused by early disc degeneration, single level Randomized to receive direct intra-disc injection of saline (n= 20), hyaluronic acid (HA, n=20), 6 million allogeneic MPCs in hyaluronic acid carrier (6M, n=30) or 18 million allogeneic MPCs in hyaluronic acid carrier (18M, n=30) Results Reduced low back pain and improved function less opioids for pain relief, greater radiographically-determined disc stability underwent less additional surgical and non-surgical treatment interventions Allogeneic Mesenchymal Progenitor Cells & Cage SHEEP sheep C3/4/5 ACD Group 1: Absorbable cage & Gelfoam sponge only Group 2: Cage & Gelfoam sponge with MPCs with chondrogenic agent pentosan polysulfate (PPS) Group 3: Cage & Gelfoam sponge with MPCs MPCs in combination with PPS to produce cartilaginous tissue Preserve motion May offer an alternative to fusion Juvenile chondrocytes 6

17 10/21/ Year follow up -Juvenile chondrocytes implanted into degenerated lumbar spine of pigs -After 12 months cells showed high GAG and Protein content - Cells proved viable by FISH (Y-chromosome) analysis after 12 months - Juvenile chondrocytes were superior according to cell viability and GAG synthesis compared to implanted MSC Three months healthy Juv. Chondr. carrier MSC FISH fluorescence in situ proves viability 2013 In vitro juv. chondrocytes Cell injection 7

18 10/21/2015 Clinical trial Juvenile chondrocytes Treatment for single level lumbar spondylosis Phase I study, 15 patients 12 month follow up, ODI, NRS, SF-36 MRI follow up 60% improved, mostly because of reduced HIZ Scores significantly improved from baseline No complications 20% had surgery Patient follow (6-12 months) Reduced HIZ after 12 months Preop. Postop. 8

19 10/21/2015 Disc Transplantation 2007 Disc Transplantation plus NP activation with htert 2013 Total disc transplant in beagle dogs Transplants injected and cultured with NP cells NP cells & human telomerase reverse transcriptase (htert) gene-transfection DMEM/F12, no cells htert may upregulate and activate NP function NP cells or htert-loaded NP cells effectively resist degeneration of the allogenic transplants htert - loaded NP cells had a better antidegeneration effect Bone Marrow Concentrate (BMC) 2015 Harvest Expansion Weeks MSC Concentrate BMC Up to 2 hours 9

20 10/21/2015 Percutaneous Injection CFU-F; colony-forming unit-fibroblast Summary: Published Clinical Studies on Disc Regeneration - All cell-based - Researcher Trial Patients Control Follow-up (M) Outcomes Journal Meisel HJ et al. Autologous Disc Chondrocyte Transplantation (EuroDisc) 28 Microdiscectomy alone 24 ADCT with discectomy shows decreased in Eur Spine J 2006, OPDQ than discectomy. No adverse risks 2008 Haufe SMW et al. Hematopoietic Stem Cell 10 No control 12 None of the patients achieved any Stem Cells Dev. improvement of their discogenic back pain 2006 after 1 year. Yoshikawa T et al. Autologous Bone Marrow Mesenchymal Cell 2 No control 24 Improvements in the vacuum phenomenon as well as signal intensity of T2-weighed MRIs. Spine 2010 Orozco L et al. Autologous Bone Marrow Mesenchymal Cell 10 No control 12 Rapid improvement of pain and disability. Transplan-tation Disc height not recovered, but water content 2011 elevated Coric D et al. Allogenic juvenile chondrocytes (NuQu) 15 No control 12 ODI, NRS, SF-36 improved from baseline. 89% of patients show the improvement on MRI. 20% of the patients underwent reoperation JNS 2013 Berlemann et al. Injectable Biomimetic Nucleus Hydrogel 14 No control 24 Significant improvement in leg and back pain after micro-discectomy Euro Spine 2009 Ruan et al. Total Disc Replacement with Allogeneic IVD 5 No control 60 The allograft engrafted the disc space without apparent immunoreaction; all minus one disc preserved range of motion Lancet 2007 Pettine et al. Injection of autologous bone marrow concentrate cells 26 No control 12 Improvement in pain scores prominently in patients with higher CFU-F concentrations. Stem Cells 2015 Rehydration of the discs observed (n=8) Non-published ongoing or terminated clinical trials Title of Trial Design N A Study Comparing the Safety and Effectiveness of Cartilage Cell Injected Into the Lumbar Disc as Compared to a Placebo Double-blind, Randamized, Phase 2 FU (M) Treatment Allogenic juvenile chondrocytes (NuQu) in fibrin carrier. PI/ Sponsor ISTO Technologies, Inc. Status Phase II done Safety and Preliminary Efficacy Study of Mesenchymal Double-blind, Precursor Cells (MPCs, Mesoblast) in Subjects With Randomized, Phase 2 Lumbar Back Pain or 18 million MPCs Mesoblast, (Mesoblast) in a Ltd. hyaluronic acid carrier Phase II done Treatment of Degenerative Disc Disease With Double-blind, Allogenic Mesenchymal Stem Cells (MSV) (Disc_allo) Randamized, Phase 1, millions MSC in 2 ml of saline Red de Terapia Celular Ongoing Autologous Adipose Tissue Derived Mesenchymal Stem Cells Transplantation in Patient With Lumbar Intervertebral Disc Degeneration Non-randamized, open label 8 6 Autologous Adipose K-Stemcell Co Tissue derived MSCs Ltd Ongoing Adipose Cells for Degenerative Disc Disease Non-randamized, open label Adipose tissue-derived stem cells suspended in Bioheart, Inc. platelet rich plasma Ongoing Intradiscal rhgdf-5 (BMP14) for Early Stage Lumbar DDD Double-blind, Randamized, Phase 1, rhgdf-5 DePuy Spine Ongoing Intradiscal rhgdf-5 (BMP14) for Early Stage Lumbar DDD Lumbar Intradiscal PRP injections Case Series Double-blind, Randomized Controlled study Autologous NP cells from fusion, cocultured with bone al Mochida J. et marrow MSCs Lutz et. al, 72 6 Single injection of PRP HSS Ongoing Complete Intradiscal injection of PRP-releasate for the treatment of lumbar disc degeneration Case-Series 6 6 Injection of the soluble releasate isolated from clotted PRP Akeda et. al., Complete 10

21 10/21/2015 Tissue engineering Tissue Engineering in the knee; ACI/ACT Methods Multistep process cartilage harvest and cell isolation cell growth (proprietary) reimplantation Chondrocyte Transplantation (ACT) ACI/ACT commercialized by Genzyme (Carticel ) Human trials since ~1990 (reimbursed since 2002) Patient load of >10,000 by 2007 (4,000/yr currently) Revision surgery for microfracture or OATS 11

22 10/21/2015 NeoCart A phase 3 product using a patient s own cartilage cells harvested from the non-weight-bearing cartilage surface of the patient s femur Crawford et al. Am J Sports Med. Crawford et al. J Bone Joint Surg Am ACT/ACI General Results New tissue formation hyaline cartilage 40-50% fibrocartilage 40-50% Success rate 1 year 80-90% 2 year 70-80% long term 50-80% Significant extension of time to TKR (from 3 yr 15+ yr) Ear Fabrication From Printed Molds Photo Reconstruction 8 week culture 12

23 3 months 1 month 10/21/2015 In Vivo Results Acellular Cellular Reiffel et al, PLoS ONE 2013 Disc Regeneration and Tissue Engineering Annulus Fibrosus (AF) AF Scaffold (Collagen gel) Mizuno H, spine 2004 Park SH, Tissue Eng Part A 2012 Martin JT et al. Acta Biomater 2014 Bowles RD, PNAS 2011 AF cells NP cells Nucleus Pulposus (NP) Tissue Engineered NP Scaffold IVD (TE-IVD) (Alginate gel) 7 days 10 days 14 days Surrounding AF contracts over time. PNAS August 9, 2011 vol. 108 no. 32 Healthy IVD Discectomy Tissue-Engineered IVD 13

24 10/21/2015 Qualitative MRI analysis: Bio-engineered Disc Post-op 1 month 4.5 month 8 month 14

25 10/21/2015 Experimental Protocol Discectomy weeks X ray & MRI Histological analysis Solely discectomized discs (n=2) Discs implanted with TE-IVD (n=12) MRI-based analysis of nucleus pulposus (NP) Size Voxel Count Hydration T2 Relaxation Time Grunert P et al. ORS 2014, Spine 2014 Post-operative Assessment X ray T2 MRIs T2 Mapping Safranin O Adjacent Healthy 16 week 10mm 500 um Discectomy 16 week TE-IVD Implantation 4 week 16 week Histological Assessment with Safranin O staining Adjacent Disc 4-week TE-IVD 16-week TE-IVD VB Discectomy VB NP VB VB VB VB AF VB VB White and black arrows indicate NP-like and AF-like cells, respectively. VB; Vertebral Body, Bars; 100 μm 15

26 10/21/2015 In Vivo Total Disc Replacement Using Tissue Engineered Disc Implant Implanted TE-IVDs Maintain its position and integrated into the native tissue Restore disc height and physiological hydration Yielded disc-like tissues over 16 weeks Annular Defect Repair Problem with surgery: Hole in disc Reherniation relates to size of the defect up to 15% experience recurrence Progressive degeneration Sealing the hole?? Cross-linked high density collagen gel Stiffer collagen with increased equilibrium modulus Decreased hydraulic permeability Highly biocompatible, supporting cell migration and matrix rearrangement (Zhang 2011, Cross 2010, Puetzer JL 2013). Rat - Tail Needle Puncture Model 16

27 10/21/2015 Summary of Results of Annular Repair X-ray Modified Pfirmann Grande NP Voxel Count Histology Healthy disc Cross-linked collagen I 74 Uncross-linked collagen Punctured Rat-tail Needle Puncture Model Fibrous cap formation Rat-tail Needle Puncture Model Future therapy options Healthy disc Early stage: Growth factors, Platelet- Rich Plasma, Gene therapy, cells Intermediate stage: MSC or Juv. chondrocyte implantation Advanced stage: Tissue engineered disc implantation, +/- Absorbable implant, Fusion, Reconstruction 17

28 10/21/2015 Summary Promising initial clinical results Questions Growth factors Gene therapy Cells Type Indications & Timing Allogeneic Disc transplants Tissue engineering work Total disc replacement Combination with structural implants / stabilization 52 The slides below are for reference Tissue-Engineered Total Disc Replacement Tissue-engineered total disc replacement (TE-TDR) Restore Motion Mechanical damping Integrate with the native tissue Produce a disc like extracellular matrix 18

29 10/21/2015 Disc Fabrication Annulus Fibrosus (AF) AF cells AF Scaffold NP cells Nucleus Pulposus (NP) NP Scaffold implant PNAS August 9, 2011 vol. 108 no. 32 Healthy IVD Discectomy Tissue-Engineered IVD 19

30 10/21/2015 Qualitative MRI analysis: Bio-engineered Disc Post-op 1 month 4.5 month 8 month Beagle C3/4 biological disc at 4 weeks 500μm Beagle MRI 20

31 10/21/2015 Low Back Pain (LBP) & Degenerative Disc Disease (DDD) Leading cause of disability worldwide. 480,000 operations per year in US for DDD. Conventional surgery does not treat underlying pathology (degeneration). Following fusion, 21.5% require reoperations (Martin BI, 2007). Prosthetic TDR Fusion Extended Fusion Table 2: Published Large Animal In vivo Studies of Cell Therapy in Disc Regeneration Species Model Cell Type Dose Outcome Reference Canine Postnucleotomy Disc Cells 6.0x10 6 Porcine Nucleotomy Allogeneic Juvenile Chondrocytes and MSCs cells/1ml/ Disc 7-10 x10 6 / ml fibrin carrier Disc remained viable, produced ECM, better maintained disc height JC outperformed MSCs in proteoglycan synthesis at 12 months Ganey T 2003, Hohaus C 2008 Acosta 2011 Porcine Postnucleotomy Human MSCs 0.5x10 6 / Implanted human MSCs survived and Henriksson HB hydrogel carrier differentiated into disc-like cells at 6 mos Canine Postnucleotomy Autologous MSCs 1.0x10 6 /ml Stem cells MSCs led to better disc height, MRI, and histology grading at 12 weeks Hiyama A 2008 Canine Postnucleotomy Bone Marrow MSCs 10 5, 10 6, 10 7 cells The disc treated with 10 6 MSCs showed more viable cells than 10 5 and less apoptotic cells than 10 5 cells at 12 weeks. Serigano K 2010 Table 3: Published Large Animal In vivo Studies of Tissue Engineering in Disc Regeneration Species Model Construct Outcome Reference Porcine Nucleotomy Cell-scaffold made of NP cells and an injectable hyaluronan-derived polymeric substitute material Injected discs had a central NP-like region with viable chondrocytes forming matrix Revell 2007 Canine Post-nucleotomy Autologous adipose tissue derived stem and regenerative cells in hyaluronic acid carrier Disc produced matrix and resembled native disc in morphology at 12 months Ganey T 2009 Canine Total discectomy Cell-allograft IVD with allograft and NP cells transduced with htert expressing viral vector Addition of htert-loaded NP cells induced resistance to allogenic disc degeneration Xin H 2013 Canine Nucleotomy Cell-scaffold composite made of three-dimensional porous PLGA scaffolds and NP cells Ruan DK 2010 Sheep Total Discectomy Absorbable interbody cage filled with mesenchymal progenitor cell and pentosan polysulfate Production of cartilaginous tissue at 3 months Goldschlager T. Neurosurg Focus Sheep Nucleotomy Allogenic disc cells in hydrogel containing hyaluronic acid Intrinsic repair of traumatic damage occurs in and maleolyl-albumin. sheep discs at 6 mo. Benz K 2012 Porcine Post-annular Injury Autologous MSCs in either Hydrogel PhotoFix (PF) or Hyaluronic Acid (HA) Treatment group had higher T2 MRI intensities and lower degeneration. Bendtsen 2011 Porcine Partial nucleotomy Bone marrow MSCs transduced with retrovirus encoding After 3 days, persistent metabolically active Olmor GW, 2014 luciferase in albumin hydrogel implanted cells in the disc Goat Post-disc injury Bone marrow stromal cells in chondroitin sulfate-based hydrogel Significant increase in NP proteoglycan accumulation at 6 months. Zhang Y, 2011 Sheep Disc height, segmental stability, and T2- weighted MRI signal were preserved Post-chondroitinase- Human Mesenchymal Precursor cells (MPCs) suspended High dose injection improved histopathology ABC injection in hyaluronic acid scores at 3 mos., while low dose at 6 mos. Ghosh P

32 10/19/2015 State of The Art Hyun Bae, M.D. Medical Director, Director of Education Cedars Spine Center Future of Regenerative Medicine in Spine 1

33 10/19/2015 Chronic Axial Lumbar Back Pain Patient profile 12.5M Patients annually present with chronic (>6 months), low back pain in the United States Only 20% present with evidence of an easily imaged pathology or anatomic source of pain 10M U.S. patients annually present with symptoms of discogenic pain: Chronic axial low back pain (> 6 months) Referred leg pain that is less than back pain Mild to moderate disc generation at 1 or more adjacent levels No significant instability or disc height loss Minimal central canal or foraminal stenosis 24 New Zealand White Rabbits (3.5 kg) L2-3 Special needle with a stopper to control the puncture depth at 5 mm. L3-4 Control L4-5 4 weeks after the puncture Experimental group 1: Lactose injection : 10 ųl of 5% lactose 2: OP-1 injection: 100 ųg of OP-1 Animal Model: Acute Disc Injury Current Models No Symptoms DDD LBP 2

34 10/19/2015 Human Spinal Disease Human Spinal Disease Structure and Vascularity of the Disc Nucleus pulposus poorly vascularized No blood vessels penetrate the inner annulus Pressure on the nucleus is 5-15 times greater than blood pressure. It is hypothesized that the tissue is hypoxic. 3

35 10/19/2015 OP-1 Pre-Op MRI rhgdf-5 Pre-Op MRI CAL BSF Delivery System Benefits Percutaneus injection of fibrin sealant (BIOSTAT BIOLOGX ) Flows into and seals fissures Fibrin matrix Fibrin Glue Study Clinical Study Biostat System Phase III Internal disc disruptions (IDD) of lumbar intervertebral discs 15 sites, N=260 One or two level, randomized, blinded First patient in February

36 10/19/2015 MRI L4-5 Disc Degeneration, Example pt treated with Fibrin Sealant pre injection post injection follow-up wdo Fibrin Sealant Biostat Fibrin Sealant for Intervertebral Disc Repair Results of the Randomized Clinical Trial: One Site Analysis Presenter Hyun Bae, MD Demographics Randomized BioStat Saline Number of patients 20 9 % Males Age (years) % Current Smokers 30% 0 % Prior Injections 80% 100% % Prior Surgery 0 0 % 12 mo 100% 100% Study is closed Except for smoking status, Demographics were similar 5

37 VAS Low Back Pain (100 mm scale) VAS Low Back Pain (100 mm scale) PreOp 1 w 13 w 26 w VAS Low Back Pain (100 mm scale) Figure 1. Self Reported Pain (VAS) Figure 2. Disability Saline-D Saline-C Saline-B Pre Tx <=1 m 3 m 6 m 12 m 24m Stu dy B Sali ne- B Stu dy C Saline-A Biostat Saline 10/19/2015 VAS Low Back Pain 26 Weeks Biostat 26% improvement Saline 88% improvement Marginal p = INTRODUCTION Is there Clinical Improvement Associated with Saline Injection for Discogenic Low Back Pain: Comparison of RCT Outcomes Recently, several multicenter clinical trials studying the effect of biologic substances or cell-based injections on lumbar intervertebral disc repair were completed. These studies all included a placebo injection with saline as a control. These studies were randomized, double blinded, and prospective. Their intent was to investigate novel treatment options for intervertebral disc repair. The findings of these studies highlight a possible reduction in pain and disability related to the saline injection. The purpose of this analysis was to evaluate saline injection related patient reported outcomes from multiple intervertebral disc injection studies. All patients were seen at same institution. METHODS A post hoc comparison was performed using data derived from four similar studies conducted at a single site that were prospective, randomized controlled, and double-blinded. Standard across the studies (A, B, C, D), patients were only included if they had symptomatic disc disease at lumbar levels of L1 to L5/S1, had a positive provocative discography, and failed at least 3 months of nonoperative treatment. Patients (males & females) ranged from 18 to 65 years of age, and were randomized into placebo (saline) or treatment (investigational substance) intervertebral disc injection groups. Visual Analog Scale for back/buttock pain (VAS, 100-mm line, with No Pain indicated at the left of the horizontal scale and Most Severe Pain at the right end of the scale) and the Oswestry Disability Index (ODI, a Likert type scale calculating functional disability) for low back pain, along with surgeon administered physical exam were completed at pre-treatment visit (pretx), and at least at 3, 6, 12 months post injection (follow-up, 12mo). Only study B utilized the Roland-Morris Disability (24 items checklist) Questionnaire instead of the Oswestry Questionnaire. Disability percentage scores were calculated: Oswestry Disability, the sum of the section scores divided by the total possible score (50 if all sections are completed), and the resulting total was multiplied by 100. Roland Morris Disability, the sum of the checked items divided by 24 multiplied by 100 to yield a percentage score. Mixed model ANOVA was used with factors of visit-time (repeated) and treatment (grouping). For all studies, side effects and adverse events were systematically collected throughout as per clinical trial standard operating procedures at the site. Hyun W. Bae, MD 1,2, Linda E.A. Kanim, MA 1,2, Samantha R. Thordarson, 1 Noelle Provenzano, 1 Janice Kim, BA 1,2, Evish Kamrava, MD 1,2, Timothy Davis, MD 1,2, Rick Delamarter, MD 1,2 1 Spine Center, Dept. of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA; 2 Spine Research Foundation, Santa Monica, CA RESULTS Control Variables: Gender and age were controlled in the overall analysis. There was a higher percentage of males enrolled across the four studies (61.5% A, 61.5% B, 60% C, 94% D) with 74% males (37/50) in the combined analysis. Males reported slightly less VAS improvement as well as less VAS pain than females preoperatively; This difference was not significant. (Males: 66.8 mm VAS pain at pretx, 39.6 mm VAS pain at 12 months post treatment, with a 41.5% difference. Females: 81.3 mm VAS pain at pretx, 32.4 mm VAS pain with a 40.6% average difference at 12 months post treatment). Age was only related to VAS at 12 months (p< 0.04, r=0.31, 9.6% of common variability). Across the studies, by 12 months, there was average 58.5% less VAS pain for saline injected patients compared to 36.6% less pain for investigational treatment injected patients (S: 20.4 mm vs. I:37.7mm p<0.01, ANOVA controlling for age, gender, study). Additionally, across the studies there was a statistically significant main effect of decrease in VAS pain for both the investigational treatment or saline injected patients (p<0.004 at 3 months; p<0.007 at 6 months; p<0.0001, 12 months compared to pre-treatment). For Disability, saline injected patients reported significantly less Disability than investigational treatment only in Study B (6 mo, p<0.04; 12 mo p<0.05). DISCUSSION An intervertebral disc injection regimen of saline may offer patients a chance for some pain resolution, decreased disability, or may merely introduce less substance reaction than to treatment or carriers and injection trauma. Noting the 50% or greater improvement observed for saline injected patients in this study provides a potentially higher threshold and means to define the MCID for injection type intervertebral disc repair treatments. Independent from the underlying reason for the observation herein, future injection studies now have a high baseline improvement threshold. A more thorough understanding of the Saline Effect and possible mechanism of action is needed. Future directions include: Testing for Saline Effect in an independent sample, with more patients, and a longer follow-up period. Differentiate between a placebo effect and a true saline effect using sham procedures* in future clinical injection studies. Significance: Although the intent was to investigate novel treatment options for intervertebral disc repair, results from these trials have elucidated a possible saline effect of improvement in self-reported VAS pain. Saline-associated clinical improvement provides a threshold for novel treatments to improve upon! Further research is needed to determine a biologic REFERENCES 1: mechanism Mavrogonatou for E, Kletsas the saline-associated D. Differential response of improvement. nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea. J Cell Physiol Mar;227(3): : Fukui S, Iwashita N, Nitta K, Tomie H, Nosaka S. The results of percutaneous intradiscal high-pressure injection of saline in patients with extruded lumbar herniated disc: comparison with microendoscopic discectomy. Pain Med Jun;13(6): * Current Randomized Clinical Trial Designs include a sham treatment group when evaluating treatments for intervertebral disc repair. VAS Low Back Pain Study B Saline-B Study C Saline-C Saline-A Study A Saline-D Study D Carrier-D Saline-D Saline-A 20 Saline-C 10 Saline-B 0 Pre Tx <=1 m 3 m 6 m 12 m 24m 6

38 10/19/2015 Results Four RCT using saline as control 58.5% decrease in patients at 12 months treated with saline 36.6% decrease in pateints at 12 months treated with investigational drug p<0.04 Allogeneic Stem Cell Stem Therapy Cells The Future? Stem Cell Pool 7

39 10/19/2015 Bioreactor Cell Processing Commercial Viability bone marrow + antibody beads/magnet Cell Bank to Provide Off the Shelf Solution MPC Highly Expandable + Non-Immunogenic bone cartilage US Bone Repair Patent granted Orthopaedic Products Products For Eye Diseases Isolated Cells Culture-expanded Cells heart muscle pancreas Products For Cardiac Diseases + Diabetes US Composition Patent granted US Manufacturing Process Patent granted Global Use Patents filed Blood vessel 24 8

40 10/19/2015 Off-the-shelf IND clearance FDA approval Lead products Congestive heart failure Bone marrow transplantation Spinal fusion Knee osteoarthritis Long bone fracture repair Acute myocardial infarction Intervertebral disc repair Eye disease (AMD) Diabetes Neurodegenerative diseases Pre-clinical Phase II Phase III Partnered with Cephalon/Teva 25 Phase II Results Study Design Prospective, multi-center, randomized, double-blind, controlled study - Patients and radiographic evaluators blinded to treatment Follow-up: 1, 3, 6, 12, 24 & 36 months Safety Evaluations Efficacy Evaluations - Adverse Events - Radiographic Changes - Treatment Failure (Surgical & Injection Interventions) o MRI - Immunological Testing o X-ray & Stability - Blood chemistry & inflammatory markers - Lower Back and Leg Pain measured by VAS Score - Radiographic - Oswestry Disability Index (ODI) o Heterotopic ossification - SF-36 o Disc degeneration - Work Productivity & Activity Index (WPAI) - Medication usage 9

41 % Responder Rate 10/19/2015 Phase 2 Clinical Study Patient Population A prospective, multicenter, double blinded, controlled clinical study of two doses of immunoselected, cultureexpanded, nucleated, allogeneic MPCs when combined with hyaluronic acid in subjects with chronic low back pain (> 6 months) due to moderate DDD at one lumbar level from L1 to S1 and unresponsive to conservative therapy for at least 3 months (including physical therapy) and evalauted at 1, 3, 6, 12, 24 & 36 months. Inclusion Criteria DDD with back pain >6 months Failed 3 Months Non-Operative Care Patients with a modified Pfirrmann score of 3, 4, 5 or 6 With or without contained disc herniation up to a 3mm protrusion with no radiographic evidence of neurological compression. Disc height loss of <30% compared to a normal adjacent disc based upon radiographic evaluation VAS Back pain >40 ODI Score >30 Exclusion Criteria Modified Pfirrmann score of 1 & 2 or 7 & 8 Clinically significant nerve or sacroiliac joint pain. Clinically significant facet pain as determined by a diagnostic medial branch block or facet joint injection Symptomatic involvement of more than one lumbar disc level. Intact disc bulge/protrusion or focal herniation at the symptomatic level(s) > 3 mm or presence of disc extrusion or sequestration Discs with full thickness tears with free flowing contrast through the annulus fibrosis Lumbar intervertebral foraminal stenosis at the affected level(s) resulting in clinically significant spinal nerve root compression. MPC groups have a greater proportion of patients with at least a 50% improvement in back pain or minimal/no residual back pain at 12 months relative to controls 80.0% 70.0% 60.0% 50.0% Proportion of patients with 50% back pain 12 months 69.2% 61.5% p = p = % 50.0% 40.0% Proportion of patients with minimal to no back 12 months 52.0% 42.3% p = p = p = p = % 40.0% 33.3% 20.0% 18.2% 30.0% 10.0% 20.0% 50% back pain months Pooled Controls 6M MPCs 18M MPCs 0.0% Minimal Back Pain ( 12 Months Pooled Controls 6M MPCs 18M MPCs Composite Endpoint for Treatment Success 50% VAS back pain reduction; AND 15 point ODI improvement; AND no intervention at the treated level MPCs groups show sustained treatment effect relative to controls over 12 months 60.0% % Meeting Treatment Success Definition 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% 1 Month 3 Months 6 Months 12 Months Saline HA 6M MPCs 18M MPCs 10

42 Proportion of Patients Proportion of Patients 10/19/2015 MPC groups have a significantly greater proportion of patients with a 50% pain reduction with no intervention compared to 24 months 70.0% 60.0% 50.0% 40.0% 50% back pain reduction with no 24 months p = p = % p = vs. saline 47.8% p = vs. saline p = vs. saline p = vs. saline 30.0% 20.0% 18.8% 10.0% 0.0% Saline 6 million MPCs 18 million MPCs MPC groups have a significantly greater proportion of patients with clinically significant function improvement and no intervention compared to 24 months 70.0% 60.0% 50.0% 40.0% 15 point ODI improvement with no 24 months p = vs. saline p = p = p = vs. saline 56.5% p = vs. saline 60.9% p = vs. saline 30.0% 20.0% 18.8% 10.0% 0.0% Saline 6 million MPCs 18 million MPCs Conclusion Allogeneic MPCs were well tolerated Both MPC doses showed improvement relative to controls for pain and functional improvement and reduced interventions Radiographic improvement in disc motion suggests improvement in disc structure and stability Over three fold increase in the number of MPC treated patients achieving concordant pain and function treatment success at both 6 and 12 months relative to saline controls Next steps: Randomized, placebo controlled phase 3 trials comparing 6M MPCs to saline placebo 11

43 10/19/2015 Missing Link Evidence Based Efficacy Phase 3 Clinical Study Groups Patients with Moderate DDD Randomize Saline Control Treatment (n=110/study) MPC Treatments (n=220/study) 6M MPCs with HA (n=110) 6M MPCs alone (n=110) 35 Mesoblast Phase III Study 60 investigative sites across US and Australia 660 subjects 2 identical protocols: MSB-DR002 MSB-DR003 Only MSB-DR003 will undergo interim analysis Study period (subject): ~15 months Randomization scheme 1:1:1 rexlemestrocel-l + saline rexlemestrocel-l + HA Saline First patient screen target date: 05 March 2015 Last patient screen target date: 09 November

44 10/19/2015 CIRM Created by Californians in 2004 with Prop 71 to Provide 3 Billion dollars for Stem Cell Research and Clinical Applications Gazit and Bae team awarded $7.1 million from CIRM to develop stem cell treatments for osteoporosis and segmental defects Goal is to develop world's first biological treatment for compression fractures Systemic Adult Stem Cell Therapy for Osteoporosis- Related Vertebral Compression Fractures CIRM Early Translational II Award (TR ) Hyun Bae and Dan Gazit Co PIs Proposed therapy MSC Injection MSCs Regenerated Vertebra PTH 13

45 Day 1 8 Weeks 10/19/2015 Generation of Lumbar Bone Defects in Osteoporotic Rats 3D Micro CT scan Bone Regeneration in Lumbar Defects of Osteoporotic Rats (µct) Untreated control PTH only PTH + MSCs Large Animal 4x15 mm 14

46 10/19/2015 Validation of design in larger animals THANK YOU 15