Efficacy of a Human Amniotic Tissue-derived Allograft, NuCel, in Patients Undergoing Posteriolateral Lumbar Fusions for Degenerative Disc Disease
Phase: NA
Conditions: Lumbar Degenerative Disc Disease, Spinal Stenosis, Spondylolisthesis, Spondylosis, Intervertebral Disk Displacement, Intervertebral Disk Degeneration, Spinal Diseases, Bone Diseases, Musculoskeletal Diseases, Spondylolysis
Sponsor: OhioHealth

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Efficacy of human amniotic tissue-derived allograft, NuCel®, in patients undergoing 
one and two level posteriolateral lumbar fusions for degenerative disc disease 
 
Principal investigator:  Joseph A. Shehadi, MD 
Sub-investigator: Timothy Meier, MD, Janet Bay, MD 
 
 
I. INTRODUCTION 
 
Lumbar degenerative disc disease is a common disorder that may cause 
debilitating symptoms.  Although there is discrepancy in the prevalence 
estimates of the morphological changes associated with degeneration (Battié et 
al. 2004), lumbar degenerative disc disease is a major cause of chronic disability 
(Modic and Ross 2007).  Risk factors include genetic predisposition, trauma, 
impaired nutrition of the intervertebral disk, mechanical loading and aging (Battié 
et al. 2004; Modic and Ross 2007).  Patients with lumbar disc disease most 
commonly present with low back pain and radiculopathy, but many are 
asymptomatic (Modic and Ross 2007).   
 
When conservative measures such as medication and physical therapy fail, 
lumbar spinal surgery is used to treat degenerative disc disease.  Spinal fusion 
surgery limits movement of the vertebrae adjacent to the affected intervertebral 
disc.  It typically consists of spinal instrumentation to secure the vertebrae, and 
bone graft material, to allow the vertebrae to fuse together (Rutherford et al. 
2007).   
 
One of the major controversies surrounding lumbar spine fusion is the choice of 
ideal graft material used to facilitate fusion.  Commonly studied properties of graft 
materials include osteoconduction (scaffolding for bone re-growth), 
osteoinduction (growth factors that induce bone growth), and osteogenesis (living 
cells that produce new bone); (Vaccaro 2002).  An ideal allograft material is one 
that is safe, effective in forming bone, forms bone where intended without 
excessive bone growth, can integrate with the host, and is cost effective.    
 
Autogenous bone graft, obtained from the iliac crest, has traditionally been 
considered the gold standard.  The advantages of an autogenous graft include 
high fusion rates, good incorporation, predictable results, osteoconduction, 
osteoinduction, osteogenesis, lack of immunogenicity, circumvents risk of 
disease transmission from another donor, and is cost-effective. (Arrington et al. 
1996; Chau and Mobbs 2009).  However, iliac crest harvest is associated with 
morbidity at the harvesting site.  Complications include pain, numbness, injury to 
nerves or arteries, infection, seromas, hematomas, peritoneal perforation, 
herniation of abdominal contents, bone fracture, and cosmetic concerns about 
the scar (Kurz et al. 1989; Arrington et al. 1996; Schwartz et al. 2009).  Post-
operative pain may lead to a longer hospital stay, and/or possibly lead to difficulty 
performing basic daily activities (Dusseldorp and Mobbs 2009; Schwartz et al. 

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2009).  Other drawbacks include limited quantity and quality of bone graft, 
increased blood loss and increased operating time (Chau and Mobbs 2009).   
 
There are several commercially available bone graft substitutes with differing 
properties.  Allograft is bone obtained from cadaveric sources, which has been 
treated to decrease its immunogenicity and risk of disease transmission.  It is 
osteoconductive, minimally osteoinductive, slower to incorporate and produces 
more variable results than autogenous bone graft (Vaccaro 2002; Miyazaki and 
Tsumura 2009).  Ceramics have been used in spinal fusions, as they are 
osteoconductive, available in large quantities and are sterile.  However, they are 
brittle and can withstand little force (Vaccaro 2002; Miyazaki and Tsumura 2009).  
Demineralized bone matrix (DBM) is both osteoconductive and osteoinductive.  It 
is produced from cortical allograft bone that has undergone acid extraction, 
removing its mineral component, but retains many proteins, including growth 
factors (Miyazaki and Tsumura 2009).  Osteoinductive growth factors have also 
been used as bone graft substitutes, and the most studied and used factor is 
recombinant human bone morphogenetic protein-2 (rhBMP-2).  Since its 
introduction commercially in 2002, rhBMP-2 has shown rapid and robust bone 
fusion, and gained widespread use, despite its high cost.  However, reports of 
serious complications have emerged, including ectopic bone formation, infection, 
urogenital events, carcinogenicity and others (Carragee et al. 2011).  This lead to 
the Yale University Open Data access (YODA) Project to re-analyze all the 
clinical data gathered by the manufacturer of rhBMP-2 (Infuse; Medtronic).  The 
results were published in June 2013, in a special edition of the Annals of Internal 
Medicine.  Two independent groups (Fu et al. 2013; Simmonds et al. 2013) found 
that rhBMP-2 was not better than autogenous bone graft in terms of fusion, pain 
or function, but posed an increased risk of adverse events.  Other emerging 
options for bone graft substitutes include autologous platelet concentrate, 
mesenchymal stem cells and gene therapy (Miyazaki and Tsumura 2009).   
 
Therapeutic use of amniotic fluid and the amniotic membrane has existed in 
medicine for decades in many different capacities (Shimberg 1938).  Some 
applications include healing of the eye, soft-tissue wounds, and bone fractures 
(Karaçal et al. 2005; Kerimoğlu 2008).  Recently, amniotic epithelial cells have 
been shown to express stem cell markers of pluripotency, including octamer-
binding protein 4 (Oct-4) and Nanog (Miki and Strom 2006; Miki 2011).  Cells 
isolated from the amnion are able to differentiate into all three germ layers: 
endoderm, mesoderm and ectoderm (Toda et al. 2007).   
 
Amniotic fluid and membrane contain mesenchymal stem cells (MSCs), which 
may be useful in graft material for bone fusion for several reasons. They have 
been shown to have osteoinductive and osteogenic properties, and therefore can 
regenerate bone.  They have been shown to selectively adhere to bone substrate 
and are unlikely to be rejected since they are immune privileged (they do not 
express human leukocyte antigen (HLA) class II molecules).  Also, stem cells 
from amniotic origin have low risk of tumorigenicity (Siegel et al. 2007).  Finally, 

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stem cells from an amniotic source do not pose ethical issues, as there is no risk 
to the mother or fetus.   
 
NuCel® is a commercially available (NuTech Medical, Birmingham, AL) source of 
MSCs derived from human amniotic fluid in combination with morselized amniotic 
membrane tissue.  In addition to stem cells, this tissue contains collagen, 
proteoglycans, hyaluronic acid, trophic proteins including bone morphogenetic 
proteins (BMPs), and growth factors.  
 
Therefore, the goal of the proposed research is to use NuCel® in the bone graft 
material applied to patients undergoing posteriolateral lumbar spine fusion, to 
assess its ability to promote bone fusion.  Its safety and efficacy will be compared 
to a standard allograft material routinely used for this surgery.   
 
 
 
II. METHODS 
 
A. Study Population 
 
Participants will consist of patients presenting with symptomatic, one or two-level 
lumbar degenerative disc disease, spondylosis or spondylolisthesis.  They must 
have failed traditional conservative treatment measures, such as pain 
medication, physical therapy, lumbar epidural steroid injections or lumbar bracing 
support, prior to surgery. They must be candidates for a one or two-level 
posteriolateral lumbar fusion, with instrumentation, with or without laminectomy.  
The participants are patients that the surgeon would consider as low risk for non-
union, and would have received only demineralized bone matrix (DBM) bone 
graft material without any other graft material, in particular, no recombinant 
human bone morphogenetic protein (rhBMP-2). Patients who would be 
considered higher risk of non-union, and who would have typically received 
rhBMP-2 will be excluded according to the list below.  
 
Inclusion Criteria 
 
 
Between the ages of 18 and 75 years 
 
Symptomatic, one or two-level degenerative lumbar disc 
disease, spondylosis or spondylolisthesis 
 
Failed conservative treatments 
 
Must be candidates for one or two-level, posteriolateral 
lumbar spine fusion 
 
Must be able and willing to give Informed Consent 
 
English-speaking 
 
 
 

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Exclusion Criteria 
 
 Smoker (any smoking ≤3 months prior to consent); (Patel et 
al. 2013) 
 Patients with poorly controlled diabetes mellitus (HgbA1c > 
7%) 
 Documented osteoporosis 
 Prior lumbar spinal surgery at the same spinal level, or 
immediately adjacent spine level, to the level being operated 
on 
 Back pain due to infection, tumour, or metabolic bone 
disease 
 Terminal disease, such as HIV infection, neoplasm  
 Autoimmune disease, such as rheumatoid arthritis 
 Morbid obesity (body mass index (BMI) of 35 kg/m2) 
 Major psychiatric illness in the last year 
 History of alcohol or drug abuse in the last year 
 Pregnant women 
 
 
 
B. Hypothesis 
 
We hypothesize that the NuCel® group will report, on average, Oswestry 
Disability Index (ODI) scores 10% lower than those of the DBM group.  Assuming 
a baseline ODI score of 52 (SD=8) for the DBM group, 90% power and an a Type 
1 error rate of 5%, a sample size of 42 patients (21 per group) would be sufficient 
to detect a 10% difference between groups using a repeated measures design 
with 6 measurement points.  To account for drop-out, loss to follow-up, and/or 
refusal to complete study documents at follow-up, we plan to recruit a total of 60 
patients (30 per treatment arm).  
 
 
C. Study Variables 
 
Clinical outcomes 
 
The following data will be collected at the time of enrollment (pre-operatively), 
and at 1, 2, 3, 6 and 12 months after surgery:  0-10 numeric pain rating scale for 
low back and leg pain, Oswestry Disability Index (ODI), and a neurological exam 
(sensory and motor evaluation).  We will also record any adverse events 
(infection, inflammation, radiculopathy, etc.) that occur intra-operatively through 
12 months.  Demographic information including gender, age, diagnosis, spinal 
level for surgery, will be recorded directly into an Excel spreadsheet for each 
patient.   
 

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Radiological outcomes 
 
Radiographs of the lumbar spine will be assessed at the time of surgery (intra-
operatively) and at 1 and 2 months (anterior-posterior and lateral) and at 3 
months (flexion-extension) after surgery to detect any spinal instrumentation 
failure.  CT scans of the lumbar spine will be taken at 6 and 12 months post 
surgery to assess fusion.  Assessment will be performed by an independent 
radiologist who will be blinded to the treatment and clinical condition of the 
participants.  The radiologist will score fusion according to the following 
categories: no fusion, any fusion, or solid fusion.  
 
The table below shows the time line for each data collection activity: 
 
 
 
 
Post-op months (range in days) 
 
Pre-
op 
Intra-
op 
1  
(25-
35 
days 
2 
(55-65 
days) 
3 
(85-95 
days) 
6 
(170-
190 
days) 
12 
(355-
375 
days) 
Oswestry 
Disability Index 
(ODI)1 
X 
 
 
X 
X 
X 
X 
X 
Pain rating 
scales 1 
X 
 
X 
X 
X 
X 
X 
Neurological 
exam1 
X 
 
X 
X 
X 
X 
X 
CT scan 1 
 
 
 
 
 
X 
X 
X-ray 2 
 
X 
X 
X 
X 
 
 
Adverse events1  
 
X 
X 
X 
X 
X 
X 
1Required for research purposes 
2Standard of care procedure 
 
 
D. Study Design 
 
The proposed study is a single-blinded randomized controlled trial.  Participants 
will be recruited from patients at Dr. Shehadi’s neurosurgery practice, 
Neurosurgery Associates, L.L.C. (Columbus, OH) or at Neurological Associates, 
Inc at Riverside Methodist Hospital with Dr. Bay.  Patients presenting with 
symptomatic, lumbar degenerative disc disease, and that meet the 
inclusion/exclusion criteria will be given information about the study and asked if 
they would like to participate. 
 
Participants will be allocated to treatment groups using a block randomization 
scheme to increase the likelihood of balance.  Treatment assignments will be 
stored in sealed, numbered envelopes that will be opened by study personnel in 

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the pre-operative period.  The patient will be blinded to treatment assignment 
throughout the course of the study.   
 
Participants will receive either standard DBM or experimental NuCel®, and will 
be blinded to which intervention they receive.  All operations will be performed by 
Dr. Shehadi or Dr. Bay. Instrumentation will consist of titanium pedicle screws 
and rods. All operations will be done according to standard methods under 
fluoroscopic guidance and with electrophysiological monitoring.  
 
Allografts  
 
DBM bone graft substitute (DBX; DePuy Synthes, West Chester, PA, USA) will 
be obtained via the Musculoskeletal Transplant Foundation (MTF), according to 
standard protocol.  DBX putty consists of 93% demineralized bone content in a 
sodium hyaluronate carrier.   
 
NuCel®, a stem-cell allograft, will be obtained from NuTech Medical, 
Birmingham, AL, USA.  The bioactive amniotic suspension is derived from 
amniotic fluid and morselized amnion.  Donors are pre-screened through 
questionnaires and laboratory tests.  Amniotic tissue is collected at the time of 
scheduled Cesarian-section, and processed by NuTech under aseptic conditions.  
The product is approved by the Food and Drug Administration.  The frozen 
contents of the NuCel® vial will be gently thawed, mixed with sterile saline, or the 
patient’s blood, and then mixed with a cancellous bone chip carrier material. 
 
Data will be collected by a member of the study team.  Clinical outcome data will 
be collected in the office through the use of questionnaires (time points described 
above).  Radiological data will be obtained from imaging location (time points 
described above).   
 
A member of the study team will be responsible for the handling and storage of 
data.  The identity of the participants will be kept confidential at all times.  
Participants will be given a number identifier for data entry and statistical 
purposes.  Documents containing the identity of the patient will be restricted to 
those investigators authorized to work on the study.  Further, these documents 
will be kept in a locked cabinet or safe, and stored with the study coordinator or 
other member of the study team.   
 
Sources of Bias 
 
Efforts were made in designing this study to minimize the chance of study biases.   
We attempted to minimize the effect of additional medical conditions by having 
strict exclusion criteria.  For example, patients who have any condition, such as 
diabetes, osteoporosis, or are smokers, are excluded from the study as these 
factors may have an effect on bone healing.  All efforts are made to recruit 
groups that are as homogenous as reasonably possible.  Participant bias will be 

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mitigated by blinding participants to the intervention they will receive.  For clinical 
outcome measures, evaluator bias is minimized as the data will be collected 
through objective questionnaires to be filled out by the participants (pain scales, 
ODI).  There is no need for evaluator judgment, which may be subjective, in 
recording these data.  For radiological outcome measures, an independent, 
blinded radiologist will score the scans, eliminating evaluator bias.  The only test 
that may be subject to evaluator bias is the neurological exam for sensory and 
motor deficits.  Having said that, the neurological exam will be performed by Dr. 
Shehadi, Dr. Bay, a physician assistant (PA) or a nurse practitioner (NP) who is 
highly skilled and experienced in evaluating this test.   
 
Since the only experimental intervention is at the time of surgery this study does 
not depend on patient adherence for the study intervention.  The study will, 
however, depend on patients adhering to follow-up office visits and scans.  We 
expect the “drop-out” rate will be low as most of these visits are required as 
follow-up to any surgery.  Also, the final time point for data collection is 12 
months after surgery, therefore, it is unlikely that the patient will seek surgical 
treatment elsewhere during this time frame.   
 
E. Statistical Analysis 
 
We will first compare the demographic and pre-operative clinical characteristics 
of the NuCel® and DBM groups to evaluate randomization fidelity. If any 
characteristics are found to be statistically significant between the groups, this 
will be accounted for in the analyses of the primary and secondary outcomes.  
We will describe all categorical variables as percentages and continuous 
variables with means and standard deviations. 
 
To evaluate our primary outcome we will use a linear mixed model with ODI 
score as the dependent variable and treatment group and time as independent 
variables.  Mixed models are often preferred over a two-way ANOVA because 
they allow for incomplete data from participants and do not require a strictly 
normal distribution in the dependent variable.  This model will also be used to 
evaluate the effect of treatment group on pain scores over time.  Statistical 
significance will be set at p<.05.   
 
We will report the frequency of observed fusion at the 6-month and 12-month 
follow-up periods via CT scan by treatment group.  If our sample size allows, we 
will compare the NuCel® and DBM groups on fusion rates at each time point 
using Fisher’s Exact tests.  Statistical significance will be set at p<.05. 
 
We will report the frequency of adverse events experienced by the NuCel and 
DBM groups including negative changes in neurological exam (sensory and 
motor) findings during the follow-up period. 
 
 
 

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III. REFERENCES 
 
 
Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA (1996).  
Complications of iliac crest bone grafting harvesting.  Clinical Orthopaedics and 
Related Research 329:300-309.   
 
Battié MC, Videman T, Parent E (2004).  Lumbar disc degeneration: 
epidemiology and genetic influences.  Spine (Phila Pa 1976) 29:2679-2690. 
 
Carragee EJ, Hurwitz EL, Weiner BK (2011) A critical review of recombinant 
human bone morphogenetic protein-2 trials in spinal surgery: emerging safety 
concerns and lessons learned. The Spine Journal 11:471-491. 
 
Chau AMT, Mobbs RJ (2009).  Bone graft substitutes in anterior cervical 
discectomy and fusion.  European Spine Journal 18:449-464. 
 
Dusseldorp JR, Mobbs RJ (2009).  Iliac crest reconstruction to reduce donor-site 
morbidity: technical note.  European Spine Journal 18:1386-1390. 
 
Fu R, Selph S, McDonagh M, Peterson K, Tiwari A, Chou R (2013).  
Effectiveness and harms of recombinant human bone morphogenetic protein-2 in 
spine fusion: a systematic review and meta-analysis.  Annals of Internal Medicine 
158:890-902. 
 
Karaçal N, Koşucu P, ÇobanǵĬu Ŭ, Kutlu N (2005) Effect of human amniotic fluid 
on bone healing. Journal of Surgical Research 129:283-287.   
 
Kerimoğlu S, Livaoğlu M, Sönmez B, Yuluğ E, Aynacı O, Topbas M, Yarar S 
(2008) Effects of human amniotic fluid on fracture healing in rat tibia.  Journal of 
Surgical Research 152:281-287.   
 
Kurz LT, Garfin SR, Booth RE (1989).  Harvesting autogenous iliac bone grafts.  
A review of complications and techniques.  Spine (Phila Pa 1976) 14:1324-1331.   
 
Miki T (2011) Amnion-derived stem cells: in quest of clinical applications. Stem 
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European Spine Journal 18:783-799. 
 
Modic MT and Ross JS (2007). Lumbar Degenerative Disk Disease.  Radiology 
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Patel RA, Wilson RF, Patel PA, Palmer RM (2013). The effect of smoking on 
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Schwartz CE, Martha JF, Kowalski P, Wang DA, Bode R, Li L, Kim DH (2009).  
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Shimberg M (1938) The use of amniotic-fluid concentrate in orthopaedic 
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