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Cryopreserved Amniotic Membrane Improves Clinical

Outcomes Following Microdiscectomy

D. Greg Anderson, MD,*

Victor Popov, MD,

Andrew L. Raines, PhD,

and Julie O’Connell, PhD

Study Design: Prospective, randomized controlled trial.

Objective: To compare pain, physical/mental functional recov-

ery and recurrent herniation for patients following lumbar

microdiscectomy with and without the use of a cryopreserved

amniotic tissue graft.

Summary of Background Data: Although microdiscectomy

procedures are routinely successful for patients with lumbar

radiculopathy due to herniated disc disease, residual low back

pain, and recurrent herniation remain unsolved clinical prob-

lems.

Methods: Following Investigated Review Board approval, 80

subjects were randomized in a 1:1 ratio to either receive cry-

opreserved amniotic (cAM) tissue or no tissue following elective

lumbar microdiscectomy surgery. cAM grafts were applied to

the annular defect at the conclusion of the procedure. Patients

provided preoperative and postoperative clinical assessment

data out to 24 months using the Oswestry Disability Index

(ODI), Short Form-12 (SF-12) Health Survey, and Visual An-

alog Pain Scale for back and leg pain. Patients with symptom-

atic recurrent disc herniation were recorded.

Results: In total, 48 males and 32 females with an average age of

47.2 years were included. Mean ODI scores for subjects treated

with cAM graft demonstrated statistically greater improvement at

6 weeks (14.49 vs. 21.82; P= 0.05) and 24 months (6.62 vs. 14.40;

P= 0.02) compared with controls. Similarly, SF-12 Physical

Component Scores demonstrated statistically greater gains in the

cAM group at both the 6 weeks and 24 months. None of the

subjects in the cAM graft group sustained a recurrent herniation

at the same surgical level, whereas 3 patients in the control group

sustained a recurrent herniation at the same surgical level, with

2 requiring fusion to manage persistent pain.

Conclusions: The data demonstrate statistically superior clinical

outcomes following lumbar microdiscectomy as measured by

ODI and SF-12 (physical composite scale) and a lower rate of

recurrent herniation with the use of a cAM tissue graft

compared with traditional microdiscectomy.

Key Words: amniotic membrane, microdiscectomy, outcome

(Clin Spine Surg 2017;00:000–000)

Lumbar radiculopathy secondary to lumbar disc herniation

(LDH) is a common health problem that presents a large

economic burden to the medical system.1,2 Nonsurgical treat-

ment options for LDH include activity limitations, pharma-

cologic therapy, manipulation, physical therapy; and epidural

steroid injections.3Although nonsurgical treatments are often

successful, patients who are nonresponsive to conservative

therapies or those with progressive neurological symptoms

require surgical intervention.

Surgical treatment is generally successful in reliev-

ing or reducing radicular symptoms due to LDH.4,5

Unfortunately, with longer-term follow-up, residual/

recurrent axial back pain and/or recurrent disc herniation

with radiculopathy are signiﬁcant unsolved problems,

with studies reporting reoperation rates following surgery

for LDH from 18.5%–25%.4,6 At present, there are

no proven treatments available to reduce the incidence

of postoperative axial back pain and recurrent disc

herniation.

The amniotic membrane (AM) is a placental-derived

tissue, sharing the same cellular origin as the developing fetus.

A primary function of the AM is to protect the fetus from the

maternal immune system and not surprisingly, the AM has

been demonstrated to possess potent anti-inﬂammatory

properties,7including the ability to reduce proinﬂammatory

and increase anti-inﬂammatory cytokine levels8and induce

apoptosis of proinﬂammatory cells.8,9 AM tissues have been

used clinically in a number of applications in ophthalmology,10

as well as a dressing for burns, nonhealing skin ulcers, and as

anaidtopromotewoundhealing.

11–13 In addition to its anti-

inﬂammatory properties, AM tissues have also been shown

to possess antiscarring properties.14,15 The anti-inﬂammatory,

regenerative and antiscarring activities of AM tissues have

recently been shown to be attributable to a unique glycoprotein

complex within the extracellular matrix called the HC-HA/

PTX3 complex.7

Received for publication April 27, 2016; accepted April 17, 2017.

From the *Department of Orthopaedic Surgery and Neurological Surgery,

Thomas Jeﬀerson University; wRothman Institute, Philadelphia, PA; and

zAmniox Medical Inc., Atlanta, GA.

The study was partially funded by a grant for clinical research from

Amniox Medical Inc.

The authors declare no conﬂict of interest.

Reprints: D. Greg Anderson, MD, Departments of Orthopaedic Surgery and

Neurological Surgery Thomas Jeﬀerson University and Rothman

Institute, 925 Chestnut St., 5th Floor, Philadelphia, PA 19107

(e-mail: davidgreganderson@comcast.net).

PRIMARY RESEARCH

Clin Spine Surg Volume 00, Number 00, ’’ 2017 www.clinicalspinesurgery.com |1

One of the hallmarks of disc degeneration is an

increase in the expression and levels of various proin-

ﬂammatory cytokines.16,17 The anti-inﬂammatory and

antiscarring actions of AM tissues make this tissue a

potentially attractive option to reduce postoperative

inﬂammation and promote healing of the annular defect

following microdiscectomy.

In the present study, we sought to determine the

safety and clinical eﬃcacy of a cryopreserved amniotic

(cAM) tissue product as an adjunctive treatment applied

to the annular defect following lumbar microdiscectomy

surgery. Speciﬁcally, we set out to compare in a pro-

spective, randomized manner, the clinical outcomes and

symptomatic recurrence rates for patients treated with

and without cAM applied to the annular defect following

lumbar microdiscectomy surgery.

METHODS

Study Design

A single-center, single-blind, prospective, random-

ized controlled trial was conducted to determine the

safety and eﬀectiveness of cryopreserved AM (cAM;

Amniox Medical Inc., Atlanta, GA) after lumbar micro-

discectomy. Because of the placement of cAM after mi-

crodiscectomy, only the patient could be blinded to

treatment assignment. The cAM tissue used in the study is

classiﬁed by the Food & Drug Administration as a human

cell, tissue, and human cellular and tissue product. Tissue

collection, processing, and use were followed according to

all applicable Food & Drug Administration regulations

and guidelines. Following Investigated Review Board

approval (Western Investigated Review Board Protocol

#20110656), enough patients were screened to be able to

enroll 80 patients randomized in a 1:1 ratio (treatment:-

control) to either receive cAM tissue (treatment) or no

tissue (control) following elective lumbar micro-

discectomy surgery. The primary exclusion criteria for the

study were a history of prior back surgery at the same

level as the herniation; inability to walk independently;

receipt of corticosteroids, radiation therapy, chemo-

therapy, or immunosuppressive agents within 1 month of

surgery; pregnancy, body mass index >50, severe renal

failure, hepatic insuﬃciency, cirrhosis; anemia, rheuma-

toid arthritis; or active local or systemic malignancy. Disc

herniations were diagnosed by combining clinical evalu-

ation and magnetic resonance imaging scanning of the

lumbar spine. All patients were treated by the same sur-

geon (D.G.A.). Patients randomized to the treatment arm

of the study received placement of cAM in the annular

defect after removal of the herniated disc fragments.

Operative Technique

All patients enrolled in the study underwent elective

lumbar microdiscectomy for symptoms of severe radicul-

opathy, unresponsive to nonoperative treatment. Brieﬂy,

after the induction of anesthesia, patients were placed

prone on a radiolucent spine frame. The incision site was

localized ﬂuoroscopically using an 18-G spinal needle. An

B20-mm incision was made on the symptomatic side and

then serial dilation was used to place a tubular retractor

(METRx, Medtronic Spine, Memphis, TN) at the aﬀected

level. A laminotomy was performed by removing the

caudal edge of the proximal lamina and the cranial edge of

the distal lamina and intervening ligamentum ﬂavum as

required to gain access the disc fragment. The traversing

nerve root was protected and retracted as necessary to

reach the herniated disc fragment. The membrane over the

extruded disc fragment was opened using a Penﬁeld #4

dissector and the herniated disc material was removed using

a pituitary rongeur. The annular defect was identiﬁed and

explored to exclude or remove additional loose disc frag-

ments. After all loose disc fragments had been removed and

the nerve root adequately decompressed, cAM was placed

into the annular defect in the cohort of patients random-

izing to cAM treatment. In the cAM cohort, a 22cm

rectangular piece of cAM was placed into the annular de-

fect. Delivery of the cAM to the annular defect was

accomplished by placing the tissue on the back side of the

nerve root retractor and the pushing the tissue into the

annular defect with a nerve hook (Fig. 1). The cAM tissue

is a thin membrane that is inserted into the void after

removal of the herniated disc material without any physical

attachment to retain the graft in position. An important

technical pearl is to reduce the intensity of suction during

delivery of the cAM tissue to prevent suctioning the tissue

up into the suction catheter. After placement of the cAM,

the nerve root retractor was removed and the tubular

retractor was withdrawn. In all patients, the fascia and skin

were closed using 2-0 absorbable suture followed by a

subcutaneous injection of 10 mL of 0.25% marcaine

(Baxter, Atlanta, GA). Patients randomized to the control

arm of the study underwent the same procedure without the

placementofcAM.

Postoperatively, patients were encouraged to begin

a walking program of at least 30 minutes per day and

all patients were referred for postoperative rehabilitation

by a physical therapist to improve core muscle strength-

ening and aerobic conditioning at 2-week postoperative

timepoint.

Outcomes Measures

Outcome instruments utilized in this study included

the Oswestry Disability Index (ODI), 10 cm Visual Ana-

log Pain Scale (VAS) (separately, for low back and leg

pain), and the Short Form-12 (SF-12) Health Survey

questionnaire. The data collection timepoints included

before surgery (baseline) and at the 2-week, 6-week,

6-month, 12-month, and 24-month postoperative timepoints.

At each in-oﬃce follow-up visit, outcome questionaires and

VAS were ﬁlled out by subjects and collected by a research

coordinator. At each out-of-oﬃce follow-up visit, subjects

were contacted by a research coordinator and provided

answers to questionaires over the phone.

Over the 24-month follow-up period, patients

were encouraged to report any symptoms of new, se-

vere back or leg pain. For patients with recurrent

Anderson et al Clin Spine Surg Volume 00, Number 00, ’’ 2017

symptoms following microdiscectomy surgery, gadoli-

dium enhance magnetic resonance imaging scanning

was utilized to diagnose recurrent LDH. Patients with

imaging evidence of disc herniation that correlated

clinically to the patient’s symptoms were identiﬁed.

Those with symptomatic disc herniation were divided

into disc herniation at the same level (characterized as

recurrent herniation) and herniation at another level

(new herniation).

Statistical Analysis

The mean ± SE for the ODI, VAS, and SF-12

scores were calculated and plotted for both the cAM and

control group at all timepoints (Microsoft Excel 2011).

Diﬀerences between groups were determined using an

unpaired ttest (SPSS Statistics, IBM, New York) with

Pr0.05 considered to be statistically signiﬁcant.

RESULTS

A total of 80 patients were enrolled in the study and

randomized in a 1:1 ratio to either the cAM cohort or the

control cohort. A summary of the patient demographics

is presented in Table 1. Brieﬂy, there were 48 males and 32

females included in the study population. The average age

was 47.2 years (range, 20–73 y). A breakdown of the

affected disc level for the entire study population is as

follows: 2 L1–L2; 4 L2–L3; 7 L3–L4; 26 L4–L5, 32 L5–

S1; 9 L4–L5, L5–S1. Overall, there were no statistically

FIGURE 1. AM tissue placement. A 22 cm piece of cryopreserved AM tissue (A). Following removal of the herniated disc,

cryopreserved AM tissue was inserted through the cannula (B) into the nucleus pulposus of the affected disc (C). AM indicates

amniotic membrane.

TABLE 1. Patient Demographics

Overall Control Amniotic Membrane

#Patients (N) 80 40 40

Age (mean) (y) 45.8 ± 11.3 47.2 ± 9.1 44.3 ± 13.1

Range 20–73 30–66 20–73

#Male 48 20 28

#Female 32 20 12

BMI 29.9 ± 6.1 28.2 ± 5.5 31.7 ± 6.3

Tobacco use 21/80 10/40 11/40

Aﬀected level

L1–L2 2 2 0

L2–L3 4 3 1

L3–L4 7 4 3

L4–L5 26 15 11

L5–S1 32 13 19

L4–L5; L5–S1 9 3 6

BMI indicates body mass index.

Clin Spine Surg Volume 00, Number 00, ’’ 2017 Cryopreserved AM Improves Clinical Outcomes

significant differences between groups in terms of sex,

age, or affected level. Postoperatively, there were no

surgical complications encountered including surgical-site

infections, nerve root injuries, or dural laceration in either

study cohort.

Scores for ODI were collected before surgery

(baseline) and at predetermined timepoints out to the

24-month postoperative timepoint. Before surgery, there

was no signiﬁcant diﬀerence in ODI scores between the

cohorts (cAM: 48.63 ± 2.69; control: 51.95 ± 2.23). At

the 2-week postoperative timepoint, nonsigniﬁcant dif-

ferences between the cAM and control group were ob-

served (cAM: 25.44 ± 3.09; control: 31.64 ± 2.80). At the

6-week postoperative timepoint, the mean ODI score for

the cAM group was signiﬁcantly lower than that of the

control group (cAM: 14.49 ± 2.63; control: 21.82 ± 2.75;

P= 0.05). Nonsigniﬁcant diﬀerences in ODI scores were

again observed between the cohorts at the 6-month

(cAM: 11.98 ± 2.50; control: 12.08 ± 2.31) and 12-month

(cAM: 9.29 ± 1.96; control: 12.83 ± 2.55) timepoints. At

the 24-month timepoint, the mean ODI scores were

signiﬁcantly lower for cAM-treated patients compared

with control patients (cAM: 6.62 ± 1.30; control:

14.40 ± 3.29; P= 0.02). Reviewing the data trends, it can

be observed that the mean ODI score for cAM cohort

continued to decrease throughout the 24-month follow-

up period, whereas the control cohort experienced its

lowest mean ODI scores at the 6-month follow-up time-

point and subsequently experienced a trend of worsening

ODI scores at the 12- and 24-month timepoints. A plot of

the ODI scores versus time is shown in Figure 2.

Mental and physical health was assessed using the

SF-12 Health Survey questionnaire. A plot of the physical

composite scores and mental composite scores is shown

in Figures 3A, B. Similar to the ODI scores, patients

receiving cAM tissue demonstrated a signiﬁcantly im-

proved mean SF-12 physical composite score at the

6 weeks (P= 0.018) and 24 months (P= 0.05) post-

operative timepoints in comparison with the control

cohort. Nonsigniﬁcant diﬀerences were observed at the

other timepoints. No signiﬁcant diﬀerences were observed

in the SF-12 mental composite scores between the

2 groups except for the 2-week timepoint where the cAM-

treated cohort demonstrated a signiﬁcant improvement

compared with the control group (P= 0.04) (Fig. 3B).

Pain was accessed using the 10 cm VAS. Signiﬁcant

improvements in both back and leg pain were observed in

both the cAM-treated and control cohorts compared with

baseline at all timepoints postoperatively. No signiﬁcant

diﬀerences in VAS scores were found between the treat-

ment cohorts (Tables 2, 3).

In the cAM cohort, there were no instances of

recurrent herniation at the same level during the

24-month follow-up period. However, in the control

cohort, 3 patients presented with a recurrent herniated

disc at the same level. All 3 patients failed to respond to a

minimum 6-week course of nonsurgical care consisting of

activity limitations, analgesic medications, nonsteroidal

anti-inﬂammatory drugs, and epidural steroid injections.

These 3 patients were ultimately treated with additional

surgery. One of the 3 (2.5% of the control cohort) had

predominantly radicular pain and was treated with

revision microdiscectomy. The other 2 patients (5% or

the control cohort) had a substantial component (> 50%

of the overall pain complex) of mechanical low back pain

and were treated with lumbar fusion.

No adverse events attributable to the cAM were

reported during the study period.

FIGURE 2. ODI. Before surgery and at specified timepoints

postoperatively, ODI scores were collected for all patients.

Values presented are mean ± SEM. cAM indicates

cryopreserved amniotic; ODI, Oswestry Disability Index.

*P< 0.05 versus control.

FIGURE 3. SF-12 Health Survey Scores. Before surgery and at

specified timepoints postoperatively, patients filled out the

SF-12 health survey. Scores for the PCS (A) and MCS (B) were

collected for all patients. Values presented are mean ± SEM.

cAM indicates cryopreserved amniotic; MCS, mental

composite scale; PCS, physical composite scale; SF-12, Short

Form-12. *P< 0.05 versus control.

Anderson et al Clin Spine Surg Volume 00, Number 00, ’’ 2017

DISCUSSION

The present investigation was performed to study the

clinical eﬃcacy and safety of a cAM tissue product placed in

the annular defect for patients undergoing routine lumbar

microdiscectomy. This prospective, randomized controlled

trial, demonstrated that cAM was able to produce better

clinical outcomes at the 6-week and 24-month timepoints as

measured by the ODI and SF-12 physical composite scores

compared with the current standard of care. In addition, in

patients treated with cAM, there were no recurrent hernia-

tions at the same level.

Recurrent disc herniation following micro-

discectomy remains an unsolved problem. In a systematic

review of the microdiscectomy literature between 2009

and 2015, the rate of recurrent herniation varied from as

little as 0.2% to 20%,18 although most of the 57 articles

reviewed reported recurrence rates between 2%–10%. In

the present study, the rate of recurrent disc herniation in

the control group (7.5%) was consistent with prior

studies, whereas patients receiving cAM did not experi-

ence any recurrent herniation at the same level (0%). This

finding is potentially quite significant and deserves more

investigation using larger patient cohorts to confirm these

early observations.

The AM has been used clinically for over a cen-

tury19 in a number of applications in ophthalmology8and

wound healing.20–22 The clinical success of cAM as a

potent anti-inﬂammatory and antiscarring agent has

recently expanded interest into the potential applications

of this unique tissue form in reconstructive procedures

where inﬂammation and adhesion formation might be

harmful. Clinical protocols have been studied for

tendon23 and nerve repair24–26 and investigations are

ongoing for various spinal applications.27

A well-characterized hallmark of disc degeneration

is the increased expression of proinﬂammatory cytokines,

including Interleukin-1b, resulted on activation normal

T-cell expressed, tumor necrosis factor-a, and substance

P.24,25 These proinﬂammatory cytokines likely play a role

in the clinical pain syndromes that require medical inter-

vention. cAM has been shown to downregulate proin-

ﬂammatory and upregulate anti-inﬂammatory cytokine

signaling.26 In addition, cAM has been shown to decrease

adhesion and proliferation of proinﬂammatory cells and

induce selective apoptosis of proinﬂammatory cells.26,27

cAM also downregulates TGF-b1 signaling, an important

component in the pathway of scar formation following

injury.13,28 This tissue acts to limit the diﬀerentiation of

ﬁbroblasts into myoﬁbroblasts to prevent collagen con-

traction, the mechanism responsible for inducing scar and

adhesion formation.29,30 AM has been investigated as a

material to reduce epidural adhesions after laminectomy

in both rat31 and canine32 models. These studies docu-

mented a reduced volume and density of scar tissue, less

inﬂammatory cell inﬁltration, and reduced ﬁbroblast

proliferation in animals treated with AM.

The immunomodulatory and antiscarring actions of

cAM tissue are modulated by a unique component of the

extracellular matrix called the HC-HA/PTX3 complex.

This complex is formed by tight association between

pentraxin 3 (PTX3) and HC-HA, which consists of high

molecular weight hyaluronic acid (HA) covalently linked

to heavy chain 1 of inter-a-trypsin inhibitor (IaI) through

the catalytic action of tumor necrosis factor-stimulated

gene-6 (TSG-6).33,34

The potential mechanisms of the beneﬁts seen in the

current study for patients treated with cAM could theo-

retically be due to the down-regulation of proin-

ﬂammatory cytokines, proinﬂammatory cells, and/or

inhibition of the scar formation pathway resulting in an

enviroment more conducive to healing of the interverte-

bral tissue.26–28 It is compelling to note the ongoing

clinical improvement was seen at each timepoint in the

mean ODI scores of the cAM treatment cohort compared

with the control group. Additional research to better

characterize the mechanism of action of cAM within the

annulus ﬁbrosus is certainly warranted.

As with any new therapy, the cost of the proposed

new treatment versus its beneﬁts should be evaluated.

This study was a single-center, early evauation of cAM as

an adjunct in lumbar microdiscectomy, and was not de-

signed to have an economic endpoint. However, the lack

of recurrent herniation in the cAM group compared with

the control group may lead to a cost-savings by reducing

oﬃce visits, medications, and repeat or additional surgical

procedures. The potential ability of cAM to reduce

TABLE 2. VAS Back Pain Data Summary

Amniotic Membrane

Mean ODI ± SE

% Decrease

From Baseline P

Baseline 5.72 ± 0.33 — —

2 wk 3.31 ± 0.48 42.1 < 0.001

6 wk 2.17 ± 0.41 62.1 < 0.001

6 mo 1.56 ± 0.37 72.7 < 0.001

Control

Baseline 5.77 ± 0.33 — < 0.001

2 wk 3.71 ± 0.46 35.7 < 0.001

6 wk 2.97 ± 0.45 48.5 < 0.001

6 mo 1.73 ± 0.47 70.0 < 0.001

ODI indicates Oswestry Disability Index; VAS, Visual Analog Pain Scale.

TABLE 3. VAS Leg Pain Data Summary

Amniotic Membrane

Mean ODI ± SE

% Decrease

From Baseline P

Baseline 8.19 ± 0.24 — —

2 wk 3.54 ± 0.46 56.8 < 0.001

6 wk 2.68 ± 0.47 67.3 < 0.001

6 mo 1.66 ± 0.40 79.7 < 0.001

Control

Baseline 8.14 ± 0.18 < 0.001

2 wk 3.82 ± 0.50 53.1 < 0.001

6 wk 3.09 ± 0.56 62.0 < 0.001

6 mo 1.81 ± 0.54 77.8 < 0.001

ODI indicates Oswestry Disability Index; VAS, Visual Analog Pain Scale.

Clin Spine Surg Volume 00, Number 00, ’’ 2017 Cryopreserved AM Improves Clinical Outcomes

overall costs associated with lumbar microdiscectomy and

recurrent low back pain in particular, should be evaluated

in a larger, multicenter clinical study.

Certain limitations of the current study should be

acknowledged. First, a relatively small number of patients

were enrolled although this deﬁciency is partially oﬀset by

the prospective randomization strategy of the current

study design. Second, all operations were performed by a

single investigator although the surgical technique was

standardized and commonplace for this type of pathol-

ogy. Third, the 2-year follow-up period represents a

medium-term outcome report. For this reason, we have

submitted an amended protocol to continue the current

research follow-up out to the 5 year timepoint to assess

longer-term diﬀerences between the cohorts.

CONCLUSIONS

Cryopreserved AM placed in the annular defect

following microdiscectomy led to improved clinical

outcome as measured by the ODI and SF-12 Physical

Component Scale at the 6-week and 24-month timepoints

compared with standard microdiscectomy. In this study,

there were no reported recurrent disc herniations at the

same level in the cohort treated with cAM compared with

standard microdiscectomy (control) which saw a 7.5%

rate of recurrent disc herniation. Overall, these results are

intriguing and further, larger studies are warranted.

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Anderson et al Clin Spine Surg Volume 00, Number 00, ’’ 2017

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