10/12/2015

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Biological Disc Regeneration and

Repair in the SpinePhysiology 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

Pfirrmann CS, Boos N, et al: Magnetic resonance imaging classification of

lumbar intervertebral disc degeneration. Spine 26:1873-78,2001

Grade 1 Grade 2 Grade 3 Grade 4 Grade 5

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IVD

Annulus Fibrosus• Outer fibrous structure with:

– (A) Fibroblast-type cells

• Scant cells

– (B) Type I collagen (abundant)

• Tensile strength.

Nucleus Pulposus• Inner gelatinous structure

– (A) Chondrocytic cells

• Proteogylcans

– (B) Type II collagen

• Compressive forces.

• Fibro-cartilaginous structure.

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

hold pos-charged H2O molecules.

– (b) Type II collagen: scaffold

Aggrecan

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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

IVDDEGENERATIVE 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

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Disc Repair• 3 main mechanisms:

– I. Growth factors: exogenous protein injection.

• Boost native chondrocytic cell production by up-

regulating 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.

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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. up-

regulate anabolic proteins or down-regulate

catabolic proteins), potentially long-term.

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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 life-

threatening 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.

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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

1

Cell Therapy

Donor Cells

Cell expansion

Carrier

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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 92:822-828, 2011

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Cartilage Cells for DDD

• NuQu: Juvenile cartilage cells

– Phase I: 15 pts - prospective, non-randomized.

– Phase II: 44 pts - prospective, randomized, blinded, placebo-

controlled.

• 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 6.75-13.5 million cells/cc

• Indiscal press (peak): Avg=92.4 psi (Range 60-101)

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: 5.7 3.8 2.5 (p=0.0036)

ODI: 53.3 26.9 14.3 (p<0.0001)

SF-36:35.5 43.4 29.2 (p=0.0014)

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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!

10/21/2015

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Current Trials and the Status of Tissue-Engineering Strategies

Roger Härtl, MD

Professor of NeurosurgeryDirector of Spinal Surgery

Department of NeurosurgeryWeill-Cornell Medical College

New York, NYUSA

Disclosure

• Consultant

• Funding

– Synthes

– Baxter

– BrainLab

– 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

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NPAF

EP

VB

Healthy Disc Degenerated Disc and Therapeutic Targets

Supply Demand

Metabolism Cells Structure

Tissue Engineered ConstructsBiomolecules Cells

bone

AFNP

EP Blood Vessel

VB

NPAFEP

DDD and Biological NP Repair

Tissue Engineering for Disc Regeneration

Biopsy

Scaffold

Cell cultivation Cell proliferation

Allograft transplant

Tissue engineered disc

Transplant

Cell transplant

Mechanical

Stimuli

Growth

Factors

Tissue developmentCell isolation

Pluripotent Stem

Cells

Progenitor Cells

Differentiated

Disc

Chondrocytes

NP AF AC

Genes

Biomaterial implantation

Early Intermediate Advanced

Viable

Cells

Structural

Damage

Biomolecular

Cell-based therapy

Engineered constructs

Therapeutic

Strategy

Degrees of

Degeneration

Stages of Disc Repair / Regeneration

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Cell implantation

Promising Animal Data:

• MSC

• Chondrocytes

• NP cells, etc.

Autologous Chondrocytes

Canine and clinical trail

Autologous cell from AF and NP

Cells expanded in culture, replanted after

12 weeks

Euro spine J, 2008

In canine

collected in

open

procedure

For clinical trial

collected during

discectomy for disc

herniation

2006, 2008

Clinical trail 24 month follow-up

- Cells were transplanted 12

weeks after discectomy

-112 patients enrolled,

patients were

randomized

- OLBPD, VAS, SF-36 scores

for follow up

- Follow up MRI

Eur Spine J 2006, 2008

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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

• 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

2006

SPINE Volume 35, Number 11

2010

- 2. case: Instability with

spinal canal stenosis L4/L5

- Two cases

- 1. case: adjacent

segment disease after

ALIF L4/L5

LBP, numbness right lower

leg 6 years after fusion

LBP, leg pain over

several years

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MRI outcome 2 Years

PRE-OP POST-OP 2yrsPOST-OP 2yrs PRE-OP

• 10 patients with DDD

• No respond to cons. Therapy after 6months

• BMA iliac crest, expanded, 12 month outcome

• VAS and ODI

• Follow up MRI was obtained

Patients showed improvement

- No increase of disc height

- Significant increase in signal

intensity after 1 year

2011

12 month outcome

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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

• 18 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

SHEEP

Allogeneic Mesenchymal Progenitor Cells& Cage

2010

Juvenile chondrocytes

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-Juvenile chondrocytes implanted into degenerated

lumbar spine of pigs

-After 12 months cells showed high GAG and

Protein content

1 Year follow up

- 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

2011

Three months

MSC

healthy

carrier

Juv. Chondr.

FISH fluorescence in

situ proves viability

In vitro juv. chondrocytes Cell injection

2013

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Clinical trial Juvenile chondrocytes

• Phase I study, 15 patients

• 12 month follow up, ODI, NRS, SF-36

• MRI follow up

60% improved,

mostly because

of reduced HIZ

Treatment for single level

lumbar spondylosis

Scores significantly

improved from baseline

No complications

20% had surgery

Patient follow (6-12 months)

Reduced HIZ after 12 months

Preop. Postop.

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Disc Transplantation

2007

• 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 antidegenerationeffect

Disc Transplantation plus NP activation with

hTERT2013

Bone Marrow Concentrate (BMC)

2015

HarvestExpansion

Concentrate BMC

MSC

Up to 2 hours

Weeks

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CFU-F; colony-forming unit-fibroblast

Percutaneous Injection

Researcher Trial Patients ControlFollow-up

(M)Outcomes Journal

Meisel HJ et al.Autologous Disc Chondrocyte

Transplantation (EuroDisc)28

Microdiscectomy alone

24ADCT with discectomy shows decreased in OPDQ than discectomy. No adverse risks

Eur Spine J 2006, 2008

Haufe SMW et al. Hematopoietic Stem Cell 10 No control 12None of the patients achieved any

improvement of their discogenic back pain after 1 year.

Stem Cells Dev. 2006

Yoshikawa T et al.Autologous Bone Marrow

Mesenchymal Cell2 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 Cell10 No control 12

Rapid improvement of pain and disability. Disc height not recovered, but water content

elevated

Transplan-tation 2011

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 cells26

No control 12

Improvement in pain scores prominently in patients with higher CFU-F concentrations. Rehydration of the discs observed (n=8)

Stem Cells 2015

Summary: Published Clinical Studies on Disc Regeneration - All cell-based -

Title of Trial Design NFU

(M)Treatment

PI/ Sponsor

Status

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

44 24Allogenic juvenile

chondrocytes (NuQu) in fibrin carrier.

ISTO Technologies,

Inc.Phase II done

Safety and Preliminary Efficacy Study of Mesenchymal Precursor Cells (MPCs, Mesoblast) in Subjects With Lumbar Back Pain

Double-blind, Randomized, Phase 2

100 366 or 18 million MPCs

(Mesoblast) in a hyaluronic acid carrier

Mesoblast, Ltd.

Phase II done

Treatment of Degenerative Disc Disease With Allogenic Mesenchymal Stem Cells (MSV) (Disc_allo)

Double-blind, Randamized, Phase 1,2

24 1225 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 6Autologous Adipose Tissue derived MSCs

K-Stemcell Co Ltd

Ongoing

Adipose Cells for Degenerative Disc DiseaseNon-randamized, open

label100 12

Adipose tissue-derived stem cells suspended in

platelet rich plasmaBioheart, Inc. Ongoing

Intradiscal rhGDF-5 (BMP14) for Early Stage Lumbar DDD

Double-blind, Randamized, Phase 1,2

38 36 rhGDF-5 DePuy Spine Ongoing

Intradiscal rhGDF-5 (BMP14) for Early Stage Lumbar DDD

Case Series10 24

Autologous NP cells from fusion, co-

cultured with bone marrow MSCs

Mochida J. et al

Ongoing

Lumbar Intradiscal PRP injectionsDouble-blind,

Randomized Controlled study

72 6 Single injection of PRP Lutz et. al,

HSS 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

Akedaet. al.,

Complete

Non-published ongoing or terminated clinical trials

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Tissue engineering

• Multistep process

– cartilage harvest and cell isolation

– cell growth (proprietary)

– reimplantation

Tissue Engineering in the knee; ACI/ACT Methods

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

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NeoCart

Crawford et al. Am J Sports Med.

Crawford et al. J Bone Joint Surg Am. 2012

A phase 3 product using a patient’s own cartilage cells harvested from the non-weight-bearing cartilage surface of the patient’s femur

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)

http://www.carticel.com

Ear Fabrication From Printed Molds

Photo Reconstruction 8 week culture

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In Vivo Results

Reiffel et al, PLoS ONE 2013

1 m

onth

3 m

onth

s

Acellular Cellular

Disc Regeneration and Tissue Engineering

10 days7 days 14 days

Surrounding AF contracts over time.

AF Scaffold

(Collagen gel)

NP cells

Annulus Fibrosus (AF)

Nucleus Pulposus (NP)

AF cells

NP Scaffold

(Alginate gel)

Tissue Engineered

IVD (TE-IVD)

Bowles RD, PNAS 2011

Mizuno H, spine 2004

Park SH, Tissue Eng Part A 2012

Martin JT et al. Acta Biomater 2014

∣ PNAS ∣ August 9, 2011 ∣ vol. 108 ∣ no. 32

Healthy IVD

Discectomy

Tissue-Engineered IVD

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Qualitative MRI analysis:

Bio-engineered Disc

Post-op 1 month 4.5 month 8 month

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Experimental Protocol

・Solely discectomized discs (n=2)

・Discs implanted with TE-IVD (n=12)

Size ⇔ Voxel Count

Hydration ⇔ T2 Relaxation Time

MRI-based analysis of nucleus pulposus (NP)

Grunert P et al. ORS 2014, Spine 2014

X ray & MRI

Histological analysis

2 4 8 16 weeksDiscectomy

Post-operative Assessment

Adjacent Healthy

Discectomy

10mm 500 um

X ray T2 MRIs Safranin OT2 Mapping

16 week

16 week

TE-IVD Implantation

4 week

16 week

Histological Assessment with Safranin O staining

White and black arrows indicate NP-like and AF-like cells, respectively. VB; Vertebral Body, Bars; 100 μm

VB

VB

4-week TE-IVD 16-week TE-IVDAdjacent Disc Discectomy

VBVB

VB VB

VB

VB

NP

AF

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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

• Problem with surgery:

– “Hole in disc”

– Reherniation relates to size of the defect

– up to 15% experience recurrence

– Progressive degeneration

– “Sealing the hole” ??

Annular Defect Repair

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

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74I

Healthy disc

Uncross-linked collagen

Cross-linked collagen

Punctured

X-rayModified

Pfirmann GrandeNP

Voxel Count Histology

Summary of Results of Annular Repair

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

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52

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

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

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Disc Fabrication

AF Scaffold

implant

NP cells

AF cells

Annulus Fibrosus (AF)

Nucleus Pulposus (NP)

NP

Scaffold

∣ PNAS ∣ August 9, 2011 ∣ vol. 108 ∣ no. 32

Healthy IVD

Discectomy

Tissue-Engineered IVD

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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

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• 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).

Low Back Pain (LBP) &

Degenerative Disc Disease (DDD)

Fusion

Extended

Fusion

Prosthetic

TDR

Canine Postnucleotomy Disc Cells 6.0x106

cells/1ml/ Disc Disc remained viable, produced ECM,

better maintained disc heightGaney T 2003,

Hohaus C 2008

Porcine Nucleotomy Allogeneic Juvenile Chondrocytes and

MSCs

7-10 x106 / 0.5-75ml

fibrin carrier

JC outperformed MSCs in proteoglycan synthesis at 12 months

Acosta 2011

Porcine Postnucleotomy Human MSCs 0.5x106 / hydrogel carrier

Implanted human MSCs survived and differentiated into disc-like cells at 6 mos.

Henriksson HB 2009

Canine Postnucleotomy Autologous MSCs 1.0x106 /ml Stem cells

MSCs led to better disc height,MRI, and histology grading at 12 weeks

Hiyama A 2008

Canine Postnucleotomy Bone Marrow MSCs 105 , 106, 107

cells The disc treated with 106 MSCs showed

more viable cells than 105 and less apoptotic cells than 105 cells at 12

weeks.

Serigano K 2010

Table 2: Published Large Animal In vivo Studies of Cell Therapy in Disc Regeneration

Species Model Cell Type Dose 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 inducedresistance to allogenic disc degeneration

Xin H 2013

Canine Nucleotomy Cell-scaffold composite made of three-dimensional porous PLGA scaffolds and NP cells

Disc height, segmental stability, and T2-weighted MRI signal were preserved

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

2010.

Sheep Nucleotomy Allogenic disc cells in hydrogel containing hyaluronic acid and maleolyl-albumin.

Intrinsic repair of traumatic damage occurs in sheep discs at 6 mo.

Benz K 2012

Porcine Post-annularInjury

Autologous MSCs in either HydrogelPhotoFix (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 luciferase in albumin hydrogel

After 3 days, persistent metabolically active implanted cells in the disc

Olmor GW, 2014

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 Post-chondroitinase-ABC injection

Human Mesenchymal Precursor cells (MPCs) suspended in hyaluronic acid

High dose injection improved histopathology scores at 3 mos., while low dose at 6 mos.

Ghosh P 2012

Table 3: Published Large Animal In vivo Studies of Tissue Engineering in Disc Regeneration

Species Model Construct Outcome Reference

10/19/2015

1

State of The Art

Hyun Bae, M.D.

Medical Director, Director of Education

Cedars Spine Center

Future of Regenerative Medicine in Spine

10/19/2015

2

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

L2-3

L3-4

L4-5

Control

24 New Zealand White Rabbits (3.5 kg)

Special needle with a stopper to control the puncture depth at 5 mm.

Experimental group

1: Lactose injection : 10 ųl of 5% lactose

2: OP-1 injection: 100 ųg of OP-1

4 weeks after the puncture

Animal Model: Acute Disc Injury

DDD LBP

Current Models

No Symptoms

10/19/2015

3

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.

10/19/2015

4

Pre-Op MRI

BSF 03-020

OP-1 rhGDF-5Pre-Op MRI

CAL 03-019

Delivery System Benefits

· Percutaneusinjection of fibrin sealant (BIOSTAT BIOLOGX®)

· Flows into and seals fissures

· Fibrin matrix

· 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 2010

Fibrin Glue Study

10/19/2015

5

MRI L4-5 Disc Degeneration,Example pt treated with Fibrin Sealant

pre injection post injection follow-up

11-003 wdo Fibrin Sealant

Biostat Fibrin Sealant for

Intervertebral Disc Repair

Results of the Randomized Clinical Trial:

One Site Analysis

Presenter – Hyun Bae, MD

Demographics

Except for smoking status,

Demographics were similar

BioStat Saline

Number of patients 20 9

% Males 55.3 66.7

Age (years) 38.6 44.1

% Current Smokers 30% 0

% Prior Injections 80% 100%

% Prior Surgery 0 0

% Follow-up @ 12 mo 100% 100%

Study is closed

Randomized

10/19/2015

6

VAS Low Back Pain26 Weeks

Biostat 26% improvementSaline 88% improvement

0

10

20

30

40

50

60

70

80

90

100

PreOp 1 w 13 w 26 w

VA

S L

ow

Ba

ck

Pa

in (

10

0 m

m s

ca

le)

Biostat

Saline

Marginal

p = 0.056

INTRODUCTION

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.

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).

Is there Clinical Improvement Associated with Saline Injection for Discogenic Low Back

Pain: Comparison of RCT OutcomesHyun W. Bae, MD1,2, Linda E.A. Kanim, MA1,2, Samantha R. Thordarson,1 Noelle Provenzano,1 Janice Kim, BA1,2,

Evish Kamrava, MD1,2, Timothy Davis, MD1,2, Rick Delamarter, MD1,2

1Spine Center, Dept. of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA; 2Spine Research Foundation, Santa Monica, CA

REFERENCES

1: Mavrogonatou E, Kletsas D. Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea. J Cell Physiol. 2012 Mar;227(3):1179-87.

2: 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. 2012 Jun;13(6):762-8.

* Current Randomized Clinical Trial Designs include a sham treatment group when

evaluating treatments for intervertebral disc repair.

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

mechanism for the saline-associated improvement.

Figure 2. Disability

Figure 1. Self Reported Pain (VAS)

Saline-B

Saline-C

Saline-ASaline-D

0

10

20

30

40

50

60

70

80

90

100

Pre Tx <=1 m 3 m 6 m 12 m 24m

VAS

Lo

w B

ac

k P

ain

(1

00

mm

sc

ale

)

StudyBSaline-BStudyC

VAS Low Back Pain

Saline-B

Saline-C

Saline-A

Saline-D

0

10

20

30

40

50

60

70

80

90

100

Pre Tx <=1 m 3 m 6 m 12 m 24m

VA

S L

ow

Bac

k P

ain

(1

00

mm

sc

ale

)

Study B

Saline-B

Study C

Saline-C

Saline-A

Study A

Saline-D

Study D

Carrier-D

10/19/2015

7

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 TherapyAllogeneic Stem Cells– The Future?

Stem Cell Pool

10/19/2015

8

Bioreactor Cell Processing

Commercial Viability

Cell Bank to Provide Off the Shelf Solution

24

Products ForEye Diseases

bone

cartilage

Isolated Cells

US Composition Patentgranted

Culture-expanded Cells

US Manufacturing Process Patent

granted

heart muscle

pancreas

US Bone RepairPatent granted

OrthopaedicProducts

Products ForCardiacDiseases + Diabetes

Blood vessel

MPC Highly Expandable + Non-Immunogenic

beads/magnet

bone marrow+ antibody

Global Use Patentsfiled

10/19/2015

9

25

“Off-the-shelf”

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

IND clearance

FDA approval

Partnered with Cephalon/Teva

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- Adverse Events- Treatment Failure (Surgical & Injection Interventions)- Immunological Testing- Blood chemistry & inflammatory markers- Radiographic

o Heterotopic ossificationo Disc degeneration

• Efficacy Evaluations- Radiographic Changes

o MRIo X-ray & Stability

- Lower Back and Leg Pain measured by VAS Score- Oswestry Disability Index (ODI)- SF-36- Work Productivity & Activity Index (WPAI)- Medication usage

10/19/2015

10

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.

Phase 2 Clinical Study Patient Population

A prospective, multicenter, double blinded, controlled clinical study of two doses of immunoselected, culture-expanded, 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.

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

33.3%

69.2%

61.5%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

50% back pain reduction @12 months

Proportion of patients with 50% back pain reduction @ 12 months

Pooled Controls 6M MPCs 18M MPCs

p = 0.009 p = 0.038p = 0.009 p = 0.038

18.2%

52.0%

42.3%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

Minimal Back Pain (≤20/100) @ 12 Months

Proportion of patients with minimal to no back pain @ 12 months

Pooled Controls 6M MPCs 18M MPCs

p = 0.011 p = 0.044

MPCs groups show sustained treatment effect relative to controls

over 12 months

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

1 Month 3 Months 6 Months 12 Months

% R

esp

on

der

Rat

e

% Meeting Treatment Success Definition

Saline HA 6M MPCs 18M MPCs

Composite Endpoint for Treatment Success50% VAS back pain reduction; AND 15 point ODI improvement;

AND no intervention at the treated level

10/19/2015

11

MPC groups have a significantly greater proportion of patients witha 50% pain reduction with no intervention compared to saline @ 24 months

p = 0.009 p = 0.038

p = 0.024vs. saline

p = 0.020vs. saline

18.8%

60.9%

47.8%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

Pro

po

rtio

n o

f P

atie

nts

50% back pain reduction with no intervention @ 24 months

Saline 6 million MPCs 18 million MPCs

p = 0.020vs. saline

p = 0.093vs. saline

MPC groups have a significantly greater proportion of patients with clinically significant function improvement and no intervention compared to saline @ 24 months

18.8%

56.5%60.9%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

Pro

po

rtio

n o

f Pa

tien

ts

15 point ODI improvement with no intervention @ 24 months

Saline 6 million MPCs 18 million MPCs

p = 0.009 p = 0.038

p = 0.024vs. saline

p = 0.020vs. saline

p = 0.020vs. saline

p = 0.093vs. saline

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

10/19/2015

12

Missing Link

Evidence Based

Efficacy

35

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)

Phase 3 Clinical Study Groups

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 2016

10/19/2015

13

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 (TR2-01780)

Hyun Bae and Dan Gazit

Co PIs

Proposed therapy

MSC Injection

PTH

MSCs

Regenerated Vertebra

10/19/2015

14

Generation of Lumbar Bone Defects in Osteoporotic Rats

3D Micro CT scan

Da

y 1

Untreated control

8 W

eek

s

PTH + MSCsPTH only

Bone Regeneration in Lumbar Defects of

Osteoporotic Rats (µCT)

Large Animal

4x15 mm

10/19/2015

15

Validation of design in larger animals

THANK YOU