Emerging Percutaneous Treatment of Discogenic LBP AAPMR 2015 Annual Assembly Boston, MA

Disclosures Co-investigator: Mesoblast; Spinal Restoration; ATRM/Depuy; Stryker Biotech; St Jude Medical; NIH funded LSS/ESI trial; SI Bone; Vertiflex; Halyard SIS AUC Comm; NASS Clinical Guidelines Comm Spine Section co-editor, Pain Medicine Consultant: Vertiflex; Zyga; Biobridges CAB: Mesoblast; Medtronic; Halyard

Disclosures Discussed technologies not FDA approved

Michael J DePalma, MD President, Medical Director Director, Interventional Spine Care Fellowship Virginia iSpine Physicians, PC President, Chairman, Director of Research Virginia Spine Research Institute, Inc President, Director iSpine Ingenuity, Inc

CLBP- Sources Most previous published prevalence estimates fall w/in our CI’s is c/w our findings all being similar, ours accurate.

How to Detect a Painful Disc

Annular Disruption and LBP Concordant pain correlated w/: > Gr III tear strongest predictor (Monetta 1994) CT: outer anular disruption (Chae-Jun 2005) 95% of painful discs had > Gr III tear (Derby 2005) If these stringent operational criteria are applied, the imputed false positive rate could be effectively reduced to zero or at least to an acceptable level of less than 10% in asymptomatic individuals and patients with chronic low back pain, except in patients with emotional problems, pain behavior and somatization, where it would remain high. In the latter patient population discogram results should be applied carefully in clinical decision making, particularly when considering discography as part of pre-operative evaluation for surgical fusion.

Anesthesia of Painful Annular Fissures DePalma Pain Med 2009

Anesthesia of Painful Annular Fissures DePalma Pain Med 2009

The Intervertebral Disc is a Common Source of CLBP d/t Painful Annular Fissures If so, how do we so, what are useful predictors and diagnostic tests?

Peripheral innervation of the anulus fibrosus is the presumed source of pain in symptomatic IDD Granulation tissue and neoinnervation present in symptomatic anular tears (Peng, ‘06, ‘07) Annular fissures are flanked by walls of innervated granulation tissue which is not typical for non-injured discs. Intervertebral discs are the largest avascular, aneural structures in the body. These areas of innervated granulation tissue are specific to the annular tear, as discovered during PLD which correlate to the areas of pain, and are absent in non-painful, degenerate discs and from areas remote from the tears w/in the same disc. Inflammation sensitizes nociceptors and mechanical loading of the disc becomes provocative. Chemical mediators of inflammation are elevated in both annular and nuclear tissue of painful lumbar intervertebral discs.15,16 Cyclical mechanical loading coupled with inflammatory stimuli increase prostaglandin E2 produc- tion by both nuclear and annular cells in vitro with the later causing a stronger reactivity than the former.17 Compared to asymptomatic discs, painful disrupted lumbar intervertebral discs have been shown to have higher concentrations of sen- sory fibers in both end plates and the nucleus.11,18 This high concentration of sensory fibers combined with increased levels of pro inflammatory mediators such as IL-8 and PGE2 cause hyperalgesia and pain.15,19 Mechanical overload of the disc can lead to annular injury. Annular injury catalyzes repair marked by an inflammatory response,13 influx of macrophages and mast cells, release of cytokines (IL-1,6,8, TNF-a, PGE2) and growth factors (bFGF, TFG-β) fol- lowed by proliferation and differentiation of disc cells concurrent with the ingrowth of blood vessels and nerve endings with concomitant fibrosis along the fissure.14 cells adjacent to the fissure and nuclear matrix degrade and nerve fibers within the granulation tissue express nocioceptive neurotransmitters.13 Chemical mediators of inflammation are elevated in both annular and nuclear tissue of painful lumbar intervertebral discs.15,16 Cyclical mechanical loading coupled with inflammatory stimuli increase prostaglandin E2 produc- tion by both nuclear and annular cells in vitro with the later causing a stronger reactivity than the former.17 Compared to asymptomatic discs, painful disrupted lumbar intervertebral discs have been shown to have higher concentrations of sen- sory fibers in both end plates and the nucleus.11,18 This high concentration of sensory fibers combined with increased levels of pro inflammatory mediators such as IL-8 and PGE2 cause hyperalgesia and pain.15,19 In addition, the whole process of healing the anulus fibro- sus injury, including inflammatory reaction, formation of granulation tissue, and tissue reconstruction had been observed, implying that the disc has actually been torn, and there has been a process of healing in progress.

Annular injury catalyzes repair marked by an inflammatory response,13 influx of macrophages and mast cells, release of cytokines (IL-1,6,8, TNF-a, PGE2) and growth factors (bFGF, TFG-β). The bFGF and TGF-􏰹, through each of their receptor signal transduction pathways, promote cellular proliferation and collagen synthesis of matrix cells, such as fibroblast (by stimulating synthesis of type I procollagen, formation of granulation tissue, and tissue reconstruction in the later stage of repair and vascular endothelial cells), which exert a strong effect on adjustment and control of wound and repair. So any one of these areas can be strategic targets for intradiscal biologic treatments. In fact, there were strong expressions of bFGF and TGF-􏰹1 and their receptors in the zone of granulation tissue in painful discs, and only weak expressions observed in nongranulation tissue zone. Thus, a close relationship between growth factor expressions following anular injury and subsequent inflammatory reaction, and disc degeneration following increased cell proliferation and differentiation activity and matrix synthesis is suggested, and these processes may be main pathogenetic features of disc degeneration. Macrophages are abundant in the granulation tissue flanking the fissures and secret FGF, TGF-􏰹, interleukin-1, and TNF-􏰴. These factors can mediate cell proliferation and differentiation, promote granulation tissue formation, and induce neo- vascularization. When there is an anular tear or injury, repair response activates from the vascularized outer layer of anulus to inner layer along the tear or injury. We found that growth factors, especially bFGF, were important as inducers of capillary ingrowth into the disc. They induced capillary endothelial cells to grow and extensively invade extracellular matrices to form capillaries proportional to the extent of bFGF expres- sion. Our current study suggests that with the occurrence of inflammatory reaction in the region of anular injury, the growth factors produced by the cells in inflammatory region act on the cells of disc, which are normally devoid of blood supply, promote differentiation and prolifera- tion of disc cells, and synthesis of extracellular matrices via signal transduction pathways. Courtesy N Bogduk

Can Experimental Technology Reconstitute the Disc Can Experimental Technology Reconstitute the Disc? Do these Changes Translate into Clinical Improvement? If so, how do we so, what are useful predictors and diagnostic tests?

Investigational Biologic Approaches Metabolic Agents Cellular supplementation to increase extracellular matrix synthesis Cells Proteins and drugs to metabolically enhance extracellular matrix synthesis Whats not here is gene therapy. No active human clinical trials. Scientific advances in metabolic agents, cells and tissue scaffolds have created unique opportunities for the development of novel therapeutic solutions for a variety of human pathologies. The continued success of tissue engineering, and the eventual development of true human replacement parts, will grow from the convergence of engineering and basic research advances in tissue, matrix, growth factor, stem cell, and developmental biology, as well as materials science and bio informatics. To understand the strengths and advantages of different biologic approaches, it is important to understand the clinical challenges associated with a degenerated and damaged intervertebral disc. Several companies are exploring the development of biologic approaches by enhancing the repair of pathogenic IDD defects. Anabolic agents, such as OP-1 and GDF-5, may promote tissue repair by increasing cellular differentiation and enhancing extracellular matrix synthesis. Others are attempting to promote tissue repair by supplementing the essentially acellular nucleus pulposus with stem cells or juvenile chondrocytes. Although promising animal results have been demonstrated with these approaches, their clinical application in humans may be limited by the avascular nature of the IVD and endplate sclerosis. It is likely that inadequate nutrition will exist to support the increased cell population or to support their stimulated matrix production. Spinal Restoration is the first to explore the potential benefits offered in a combination product that provides both a degradative, conductive tissue scaffold and a metabolic agent that promotes extracellular matrix accumulation by inhibiting inflammation and catabolic tissue degradation. Tissue Scaffolds Conductive scaffolds to enhance cellular migration, proliferation and extracellular matrix synthesis in three dimensions

Investigational Biologic Approaches Intradiscal Metabolic Agents Proteins and drugs to metabolically enhance extracellular matrix synthesis ADVANTAGES DISADVANTAGES Anabolic benefit (inductive) Anti-catabolic Proliferative Extensive In vitro and animal preclinical evidence Viable cell content? Limited cellular nutrition? Vertebral osteolysis? Extradiscal leakage? Expense? Anabolic, anti-catabolic, cause cells to proliferate in in vitro under ideal conditions. But can this occur w/in the disc? Do they have nutritional support and building blocks to build PG and matrix (water, carbon, collagen). Bmp’s/GF stimulates a strong inflammatory response causing local bone resorption (saw this in acdf w/ bmp- used L/S fusion dose which was too high for c-spine). Trabecular bone does not come back once you break it. Bmp’s are stored naturally in bone. w/ local resorption there is add’l release of bmps. Extradiscal leakage into foramin, epidural space, and expensive to be produced.

Investigational Biologic Approaches Intradiscal Metabolic Agents Stryker (OP-1) FDA Phase II (44) Completed (2007) Weeks after Puncture 4 8 12 16 20 Disk Height Index [%] 100 80 60 40 120 Injection 100 mg rhOP-1 [Masuda, Spine 2006] Non-puncture Puncture Rabbit In vivo data H/E stain picture published density and type of tissue produced by the GF. Did the GF stimulate natural tissue like the top slide (native control). The bottom 2 look different. This was a rabbit model w/ disc puncture induced DDD w/ ideal supporting cast (young discs w/o endplate abnormalities.

Investigational Biologic Approaches Intradiscal Metabolic Agents Depuy (GDF-5) FDA Phase I/II (30) Enrolling (2012) FDA Phase II (30) Enrolling (2013) Weeks after Puncture 4 8 12 16 20 Disk Height Index [%] 100 80 60 40 120 Injection 100 mg rhGDF-5 [Chujo, Spine 2006] Non-puncture Puncture Rabbit In vivo data Similar rabbit model. Slides show matrix (fibroblastic vs chondrocytic). These slides are more favorable but still in dosing trials and not yet published.

Investigational Biologic Approaches Intradiscal Cells Cellular supplementation to increase extracellular matrix synthesis Juvenile chondrocytes [ISTO Technologies] Meshenchymal Presursor cells [Mesoblast] Progenitor cells [Discgenics] ADVANTAGES DISADVANTAGES Juvenile chondrocytes do not induce rejection. Putting in chondrocytes to restore whats missing (matrix, PG). Stem cells have to differentiate into chondrocytes to do this too but need stimulation from proper GF (TGF B-early signal stimulating repair produced duirng inflammatory cascade so causes some tissue resorption 1,2, PDGF- occurs when matrix syn is occuring) to do so. Cell viability- injection forces are high enough to possibly injuring/killing the stem cells- shear forces are high during injection. If cells don’t differentiate to what you desire, what do they produce? Can the disc support the injected cells if the functional spinal unit contains nutritional support for a certain number of disc cells already. Anabolic benefit Anti-catabolic (stem cells) Extensive In vitro and animal preclinical evidence Cell viability? Limited cellular nutrition? Differentiation signals? ECM similarity? Expense?

Investigational Biologic Approaches Intradiscal Cells ISTO (NuQu) (Juvenile chondrocytes) FDA Phase I (15) Complete (2010) Rat - In vivo data (2008) Non-needle puncture Needle puncture and NuQu (saffrin o stain- everything orange is PG) Nucleus is filled with type II cartilage (biologic nucleoplasty), fibrosed over. Is this the response we want. But annular architecture seems to look good. This is a rat model but still favorable results. Needle puncture

Investigational Biologic Approaches Intradiscal Cells Juvenile chondrocytes (ISTO NuQu) FDA Phase I (15) Complete (2010) Swine- In vivo data Using a swine model and performing a nucleotomy via mechanical perc discectomy after stab incision, JC, fibrin carrier, or MSC were injected into disc. MSC’s were harvested from male yorkshire pigs, and expanded in culture for 12 days; JC’s were harvested from 7 day old miniature pig femur chondyle articular cartilage and then expanded in cell suspension; fibrinogen centrifuged from whole blodd from single male mini pig used as carrier. DNA, s gag, and protein content were measured at 3, 6, and 12 months after aniimal sacrice. JCs produced a type II collagen-rich matrix that was largely absent of type I collagen. Viable JCs were observed at all time points, whereas no evidence of viable MSCs was found. These data support the premise that committed chondrocytes are more appropriate for use in disc repair, as they are uniquely suited for survival in the ischemic disc microenvironment. FISH localization demonstrating the presence of donor cells at 3 months and 12 mon.s. bottom line: 1. There was unequivocal proof that allogeneic JCs injected into denucleated healthy discs remain viable and contribute directly to the production of Safranin- O positive ground substance identified in the disc space 3, 6, and 12 months post-operatively 2. Our data demonstrate that JCs are capable of surviving and producing cartilage matrix within the IVD environment. 3. No MSC’s noted at 12 mons 4. These data support the premise that committed chondrocytes are more appropriate for use in disc repair, as they are uniquely suited for survival in the ischemic disc microenvironment. Acosta Tissue Engineering 2011

Investigational Biologic Approaches Intradiscal JC- Cells- ISTO 15 patient pilot, 2 site pilot study 1 year outcome Primary endpoint= safety Secondary endpoint= effectiveness (VAS, ODI, SF-36) Single level + PLD- gr III or IV (exc. V) (Coric J Neurosurg:Spine, 2012)

Investigational Biologic Approaches Intradiscal JC- Cells- ISTO Results: 14 subjects: BL- NRS/ODI was 5.7 / 53.3% 12 mon - NRS / ODI was 3.07 (p<0.0019) / 20.2% (p<0.0001) (Coric J Neurosurg:Spine, 2012)

Investigational Biologic Approaches Intradiscal JC- Cells- ISTO

Investigational Biologic Approaches Intradiscal JC- Cells- ISTO Results: 87% (95%CI:78-96) 30% improvement in ODI 3 subjs underwent TDR No SAE’s (Coric J Neurosurg:Spine, 2012) Based on these results are larger phase II study is under way

Investigational Biologic Approaches Intradiscal Cells Mesenchymal Precursor Cells/HA (Mesoblast) FDA Phase II (100) Enrolled (2014) Sheep- In vivo data (2012) Highly purified, immunoselected Stro-1/Stro-3 positive Mesenchymal Precursor Cells (MPCs) -  Relatively homogeneous,well characterized cells -  Increased in vitro differentiation efficiency into cells of the bone, fat and cartilage lineages  Not immunogenic and suppress immune responses MPCs secrete multiple factors in the appropriate concentration, sequence and duration in response to disease/injury-specific micro- environmental cues -  Detect injury and inflammation -  Respond to local stimuli and signals from the injured tissue -  Releaseawiderangeofbiomolecules(growthfactors, chemokines, enzymes etc.) -  Increasedproteoglycansynthesis -  increasedmigration/proliferationofnucleuscells Ghosh 2012

Investigational Biologic Approaches Intradiscal Cells MPC/HA (Mesoblast ) FDA Phase II (100) Enrolled (2014) Sheep- In vivo data (2012) Previously, no one had shown matrix synthesis w/ stem cells. 1. compared to injected HA 2. discs injected with ABC which causes degeneration that can heal on its own At 3 months and 6 months after high dose stem cell injection, the mean MRI aggregate degeneration scores were significantly less than that of chondroitinase treated and HA treated discs but not different than control discs. Ghosh 2012

Investigational Biologic Approaches Intradiscal Cells (Mesoblast) Mean Disc Height Index (%) 0.5 M MSC 4.0 M MSC HA+MSC cABC HA 3 mos 6 mos PT Con * cABC concentration inadequate to stimulate chronic disc height loss. Degenerative discs spontaneously recovered HA carrier tx’d discs spontaneous disc height recovery less Addition of MSC overcame carrier limitations to match spontaneous disc height recovery * p <0.01 relative to control values # p <0.01 relative to MSC

Investigational Biologic Approaches Intradiscal Stem Cells- Mesoblast 100 patient RCT- single ID inj 3 year outcome: results @12 mon Primary endpoint= safety Secondary endpoint= effectiveness (VAS, ODI, MRI) Single level mod Pfirrmann 3-6; type I or II MC Incomplete annular tear (Gr V exc) (NASS Ann Mtg, Nov 13, 2014; DePalma SIS ASM, 2014)

Investigational Biologic Approaches Intradiscal Stem Cells- Mesoblast Results @ 12 months: 30 subj: 6M cells; 40 subj: control % who had > 50% reduction of VAS: 69% (95%CI: 53,86) tx arm 33% (95%CI: 19,48) ctrl arm % with residual < 2/10 VAS LBP: 52% (95%CI: 34,70) 18% (95%CI: 6.1,30) (NASS Ann Mtg, Nov 13, 2014, DePalma SIS ASM, 2014)

Investigational Biologic Approaches Intradiscal Stem Cells- Mesoblast The magnitudes of intervertebral rotation and translation that occurs in the sagittal plane between the flexed and extended positions are the most commonly used measures of spine motion Changes in translational motion correlate with disc stability in early disease (Pfirrmann Grade <5), as in this study population1 There were no significant differences between groups at baseline for radiographic measurements or spinal motion, suggesting that the differences at 12 months reflect treatment related changes 1 Inoue et al. Othop Clin N Am 42 (2011) 487-499 NASS Ann Mtg, Nov 13, 2014

Investigational Biologic Approaches Tissue Scaffolds Conductive scaffolds to enhance cellular migration, proliferation and extracellular matrix synthesis in three dimensions Fibrin sealant [Spinal Restoration] ADVANTAGES DISADVANTAGES Anabolic (inductive, conductive) Anti-catabolic Proliferative Extensive In vitro, animal and human preclinical evidence Inexpensive Viable cell concentration? Scaffold longevity? Biocompatibility? Combination of fibrinogen, thrombin, and aprotinin to initiate last stage of clotting cascade, and inhibits proteolytic enzymes.

Fibrin Sealant: Potential benefits for pathogenic IDD dependence Cellular Vascular dependence Natural soft tissue processes Hours Days Weeks Time Months Bleeding Fibrin sealant Clot formation Inflammation Proliferation The pathogenesis of intervertebral disc (IVD) degeneration is a complex process influenced by several poorly understood biological and mechanical factors including reduced cellular nutrition, quantitative and qualitative changes in matrix turnover, loss of cells, neurophysiologic abnormalities and altered biomechanics.1 Damaged and degenerated intervertebral disc tissues contain increased concentrations of several proinflammatory cytokines and proteolytic enzymes such as tumor necrosis factor-α (TNFα), interleukins (IL-1β, IL-6, IL-8) and matrix metalloproteinases (MMP-1,2,3). Chronically elevated concentrations of these cytokines lead to decreased proteoglycan synthesis and increased matrix catabolism. These catabolic processes have been linked to the formation and progressive accumulation of radial and concentric tears of the anulus fibrosus associated with pathogenic IDD. Normal anabolic repair of the intervertebral disc is inhibited because of its avasculature. The lack of a vascular supply prevents the deposition of a provisional fibrin scaffold that typically facilitates cellular soft tissue repair. As a novel, minimally-invasive therapeutic option (CLICK), we hypothesized that by delivering fibrin sealant to the pathogenic intervertebral disc, would provide the missing signals necessary for intervertebral cells to down regulate inflammation and re-establish normal soft tissue repair. Published studies suggest that Biostat BIOLOGX Fibrin Sealant® (FS), its components (fibrinogen, thrombin, Factor XIII, and synthetic aprotinin acetate) and its degradation products facilitate the healing of chronic wounds by reducing inflammation and stimulating cellular migration, proliferation and extracellular matrix formation.2 Discogenic pain and pathogenic disc degeneration are associated with the presence and accumulation of concentric and radial tears in the anulus fibrosus. Because the intervertebral disc is vascularized only in its periphery, these defects accumulate over time because of impaired healing. (CLICK) The lack of a vascular supply inhibits the deposition of a fibrin clot which serves as a tissue scaffold to assist in the normal sequence and strongly inter-related processes of cellular soft tissue repair. (CLICK) In the intervertebral disc, impaired healing clinically manifests in chronic inflammation. Published literature clearly implicates chronic inflammation with the nuclear accumulation of pro-inflammatory cytokines and proteolytic enzymes. These factors lead to degenerative changes in intervertebral tissue composition and structure and are associated with adjacent vertebral bone resorption and endplate sclerosis. More importantly, several pro-inflammatory cytokines commonly found in high concentrations within the nucleus pulposus are hyperalgesic and can stimulate discogenic back pain through nociceptive nerves located within the anulus fibrosus and the adjacent vertebral endplates. As a novel therapeutic option (CLICK), it is proposed that by delivering fibrin sealant to the pathogenic intervertebral disc, nature’s preferred soft tissue healing matrix could provide the missing scaffolding necessary for intervertebral cells to down regulate inflammation and re-establish normal soft tissue repair. Catabollic Tissue Resorption Anabolic Tissue Formation Catabollic Tissue Resorption Anabolic Tissue Formation Tissue Repair Tissue Remodeling

Preclinical Porcine Study Disc Histology Investigational Biologic Approaches Tissue Scaffolds Spinal Restoration FDA Phase III Enrolled (2012) 3 Week 12 week Control DEGN FS DEGN Treatment discs exhibited progressive invasion of anular fibrotic tissue into the NP FS supplementation inhibited nuclear fibrosis and stimulated PG synthesis Potential benefits of FS: Space filling, protection of NP Conductive degradable tissue scaffold Surface, cell mediated degradation Preclinical Porcine Study Disc Histology

Investigational Biologic Approaches Tissue Scaffolds Denucleated discs (DEN) Time [wks] IL-1b TNFa IL-6 IL-8 IL-4 TGFb 2,3 +129% +126% 160% +313% +33% +6% 6 +4% +114% -16% -32% +70% 12 -20% +52% +2995% +265% +49% Fibrin supplemented treatment discs (FS) Time [wks] IL-1b TNFa IL-6 IL-8 IL-4 TGFb 2,3 -19%+ -34%+ +25%+ 130%+ +204%*+ +223%*+ 6 +100% +165% +167% -58% -88% +21% 12 -45% +56% +32% +260% 0% +120% FS treated discs upregulated IL-4 and TGF-beta growth factors suggesting an anabolic effect and soft tissue growth by increasing fibroblasts and chondrocytes. Biochemical Compositional Analysis Relative cytokine synthesis [pg/ng DNA]

Investigational Biologic Approaches Tissue Scaffolds Spinal Restoration FDA Phase III Enrolling (2011) (BIOSTAT BIOLOGX) Porcine In vitro data (2011) MMP-1 MMP-2 MMP-3 100 102 104 106 108 NP NP 100 102 104 106 108 NP 100 102 104 106 108 Matrix metalloproteinases are catabolic- 1,2 degrade collagen, 3 degrades PG and FS. Again, FS blunts expression of MMP’s that are responsible for degrading PG and collagen. 4 7 14 Time [Days] 4 7 14 Time [Days] 4 7 14 Time [Days]

Investigational Biologic Approaches Tissue Scaffolds Spinal Restoration FDA Phase III Enrolling (2011) (BIOSTAT BIOLOGX) Human Phase I/II data (2011)

Investigational Biologic Approaches Tissue Scaffolds Spinal Restoration FDA Phase III Enrollment complete (2012) (BIOSTAT BIOLOGX) RCT 220 subj.s - 26 wk primary endpoint: -33.5% TX vs 39.3% CTL achieved successful outcome -42.1% TX vs 50.0% CTL had clinically meaningful reduction in LBP -52.4% TX vs 50.5% CTL had clinically meaningful reduction in RMD Spinal Restoration Press Release 7/13

Conclusions Painful discs cannot be explained solely by degeneration Painful annular fissures flanked by innervated granulation tissue Chronic LBP is related to non-healing of painful fissures

Conclusions Growth factors, cell supplementation, tissue scaffolding Combination of technologies Ideal treatment hasn’t yet been identified But must address regeneration as well as reconstitution of altered tissue mechanical properties

Thank you