Journal of Clinical Neuroscience 89 (2021) 412–421

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Journal of Clinical Neuroscience

journal homepage: www.elsevier.com/locate/jocn

Clinical study

Homologous amniotic membrane as a dural substitute in decompressive

craniectomies
Elisabetta Marton a, Enrico Giordan b,⇑, Paolo Gallinaro b, Christian Curzi a, Diletta Trojan c, Adolfo Paolin c,
Angela Guerriero d, Sabrina Rossi e, Matteo Bendini f, Pierluigi Longatti a, Giuseppe Canova b
a
Department of Neuroscience, University of Padova, Padova, Italy
b
Department of Neurosurgery, Aulss 2 Marca Trevigiana, Treviso, Italy
c
Tissue Bank Foundation (FBTV), Treviso, Italy
d
Department of Pathology, Aulss 2 Marca Trevigiana, Treviso, Italy
e
Department of Pathology, Bambino Gesù Children’s Hospital, Rome, Italy
f
Department of Radiology, Aulss 2 Marca Trevigiana, Treviso, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: A dura mater substitute in decompressive craniectomies must protect the brain while
Received 6 March 2021 providing a dissection plane between the cortex and myocutaneous layer. The human amniotic mem-
Accepted 17 May 2021 brane (AM) has anti-inflammatory, wound healing, and differentiation properties. We tested AM proper-
ties as a dural substitute by comparing the outcomes to biological ones.
Methods: We prospectively collected data on 25 patients who randomly underwent decompressive
Keywords: craniectomy with lyophilized AM patches and 25 in which biological substitutes were utilized between
Amniotic membrane
2015 and 2019. The AM was laid with the epithelial side facing the brain because of the anti-adhesive
Trauma
Neuro
proprieties, while the chorion facing the myocutaneous flap. We collected data on demographics, neuro-
Decompressive logical status, comorbidities, and surgical outcomes. Additionally, we created a score – dura mimicking
Craniectomy score– and reviewed postoperative imaging and pathological specimens.
Cranioplasty Results: The majority (96%) of AM grafts were integrated into native dura. Thirteen patients scored as
Intracranial pressure excellent and 11 good on our ‘‘dura mimicking score”, showing tissue integration ability but no cerebral
cortex adhesion. The histopathological analysis showed that AM had thick plates of dense fibrous tissue
with small reactive vessels, reactive fibroblasts, and lymphocytes infiltrate. The AM group’s first out-
comes were not different from the biological substitute patients but higher integration rate to the dura
and less adhesion to the myocutaneous flap in AM patients.
Conclusions: We documented the anti-adhesive, protective, and integrative properties of AM dural sub-
stitute patches in patients who underwent decompressive craniectomies, comparing the intraoperative
differences and postoperative outcomes to biological dural substitutes.
Ó 2021 Elsevier Ltd. All rights reserved.

1. Introduction cranioplasty. Also, to avoid CSF fistulas, watertight dura

Decompressive craniectomy is a last resort for the treatment of
intractable intracranial hypertension (ICH). It consists of decom-
pressing the brain by removing a large bone flap, along with a wide
dural opening, allowing cerebral expansion beyond the regular cra-
nial vault. The herniated brain is then covered with the native dura,
strengthened by a dural substitute patch, to separate and protect
the parenchyma from the overlying myocutaneous layer.
The primary concern is detection, splitting, and reconstruction
of every layer from scarring and adhesions during the subsequent
⇑ Corresponding author at: Aulss 2 Marca Trevigiana, Via Piazzale 1, Treviso, Italy.
E-mail address: enrico.giordan@aulss2.veneto.it (E. Giordan).
reconstruction is required.
Different tissues and materials such as fascia lata, galea/pericra-
nium, synthetic meshes, or animal-derived collagen matrix and
pericardium (i.e., bovine), have been tested as dural substitutes
in decompressive craniectomies. Dural substitutes can be artificial
(synthetic) or biological (heterologous or autologous) tissue [1].
Even the human amniotic membrane (AM) was considered for this
purpose [2,3]. AM is well renowned for its anti-inflammatory prop-
erties, which reduce the risk of scarring and adhesion formation,
and its wound healing proprieties because it is rich in growth fac-
tors that promote cell differentiation.
The ‘‘optimal” dural substitute has not been found yet. Even
biologic substitutes do not integrate optimally with the autologous

https://doi.org/10.1016/j.jocn.2021.05.030
0967-5868/Ó 2021 Elsevier Ltd. All rights reserved.
E. Marton, E. Giordan, P. Gallinaro et al.
dura layer. This could favor CSF leak formation or stimulate a
phlogistic response with consequent adherence among the dural
layer and brain cortex.
AM has been widely used in many clinical and surgical fields
(i.e., ophthalmic [4], plastic [5], ENT [6], ortopedic [7–9], and uro-
logic surgery [10–12]). AM’s favored use is strongly related to its
antiangiogenic, anti-inflammatory [13], and anti-fibrotic [14]
effects. Also, AM has antimicrobial [15,16] and strong epithelializa-
tion [17] proprieties [3,18–21].
Dural substitutes with anti-inflammatory properties might
reduce the risk of adherence formation, thus decreasing operative
time, blood loss, risk of CSF fistula formation, infections, and other
complications [22,23].
We describe our experience using lyophilized homologous AM
as a dural substitute in decompressive craniectomies. We had
two aims with this study. First, to determine if AM as a dural sub-
stitute can reduce the risk of adhesions with the parenchyma while
avoiding CSF leakage and infections by comparing patients’ ran-
domly undergoing decompressive craniectomy using AM or biolog-
ical substitutes. Second, evaluate the reconstruction process’s
feasibility during cranioplasties and evaluate AM’s ability to inte-
grate with the native dura.
2. Materials and methods
The Ethical Committee approved the study of AULSS 2 Marca
Trevigiana. We prospectively collected decompressive craniec-
tomies patients in whom lyophilized homologous AM was used
as a dural substitute and compared them to a group of prospec-
tively collected patients with decompressive craniotomies with
biological dural substitutes. Patients collection was conducted at
our Institution (AULSS 2 Marca Trevigiana, Treviso, Veneto, Italy)
between January 2015 and January 2019.
We enrolled consecutive patients who underwent decompres-
sive craniectomy and considered equally suitable for AM or syn-
thetic/biological dural substitute.
The patients who had met our inclusion criteria were given an
alphanumerical identification number and randomly assigned by
a registered nurse to the AM group and the biological dural substi-
tute group. The surgeon was aware of the patient’s group allocation
in the operating room at the time of the cranioplasty procedure
and then kept blinded to patient follow-up and data interpretation.
The authors were also blinded to the group allocation until the end
of data analysis or imaging interpretation. The MRI imaging was
conducted under the supervision of an expert neuroradiologist.
2.1. Patients selection
Patients selected for this study were younger than 70 years and
eligible for decompressive craniectomy when intracranial hyper-
tension (ICH) values were > 25 mmHg because of brain ischemia,
spontaneous intraparenchymal hematoma, or trauma. Decompres-
sion was considered when ICH was unresponsive to medical ther-
apy (barbiturate sedation, infusion of osmotic agents as mannitol /
hypersaline solution). Such occurrences were accompanied by a
neurological deterioration (2 points on Glasgow come scale
(GCS) or GCS motor component and/or anisocoria), and abnormal
computer tomography (CT) or magnetic resonance imaging (MRI)
scans (i.e., malignant edema, midline shift > 5 mm, absence of basal
cisterns and vault sulci distortion). All patients with severe neuro-
logical deterioration (GCS  4 or GCS motor component  2),
mydriatic pupils, or actual brainstem damage did not undergo
decompressive craniectomy and were excluded.
Patients who had met our inclusion criteria were given an
alphanumerical identification number and randomly assigned by
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Journal of Clinical Neuroscience 89 (2021) 412–421
a registered nurse to the AM group and the biological dural
substitute group. Biological patch (DuraformÒ Dural Graft Implant
[Codman, Jhonson& Jhonson] or TutopatchÒ [SIAD Healthcare,
Italy]) are interchangeably used in our Institution and made of a
collagen matrix derived and processed from bovine pericardium
and tendons, respectively.
Patients selected for this study had a postoperative brain CT
scan performed within 24 h after craniectomy to exclude postoper-
ative complications, a brain MRI performed 30 days after craniec-
tomy, and a late contrast-enhanced brain MRI scans performed
after cranioplasty within 6 months. The lack of neuroradiological
imaging excluded patients from the study.
2.2. Data collection
For both groups of patients, we collected data on age at decom-
pressive craniectomy (years), sex, admission diagnosis, admission
GCS, relevant comorbidities (hypertension, diabetes mellitus, or
state of immunodeficiency secondary to medications or patholo-
gies), medical treatments (i.e., immunosuppressive drugs and
blood thinners), laterality of decompression, and estimated dural
defect area (cm2).
During cranioplasty, we evaluated operative time (minutes) and
blood loss (mL).
Additionally, in the AM patients group, we designed a ‘‘dura
mimicking score” to assess AM proprieties. The ‘‘dura mimicking
score” is a simple but effective score divided into three categories:
sufficient (1), good (2), and excellent (3), based on the surgeon’s
intraoperative assessment of the AM’s adhesion to native dura,
cerebral parenchyma protection, and integrity (transparency),
thickness, and adherence to underlying cerebral parenchyma. The
score aims to compare the AM patch behavior and aspect before
cranioplasty to the native dura regarding potential substitute prop-
erties. The score is described in detail in Fig. 1. We adopted the
same ‘‘dura mimicking score” to biological dural substitute to
make a valid comparison between available options for dural
reconstruction [24–26].
Generally, integration was considered satisfactory when the
surgeon found a single tissue layer without recognizing a clear dif-
ference between native dura and AM patches and when, later at
microscopical analysis, fibroblasts and neovascularization were
found abundantly.
We also reported postoperative surgical complications within
30 days, the need for additional interventions, and time from
decompressive craniectomy to cranioplasty (days) for both groups
of patients.
In addition, we also studied postoperative imaging (i.e., AM
hyperintensity on T1-CE weighted sequences) for both groups
and histopathological findings of AM biopsied during cranioplasty.
All both arms patients underwent imaging as follow: 1) postop-
erative CT scan to highlight adverse events (within 24 h from
decompression); 2) brain MRI (Siemens, Avanto 1.5) with isovolu-
metric T1-contrast enhanced (T1-CE) and isovolumetric T2-Flair
scans after decompression surgery (between 1 and 7 days), 3)
before cranioplasty (30–40 days after decompression); and 4) after
cranioplasty (3 months after reconstruction surgery). However,
some patients underwent a tailored imaging protocol based on
the underlying pathology and clinical necessities. Isovolumetric
slice thickness was set at 1 mm. Our imaging analysis was preva-
lently focused on T1-CE sequences. Indeed, we believed that T2-
Flair sequences might overestimate AM signal as a consequence
of either hyperemia (i.e., neoangiogenesis) or, worse, postoperative
iatrogenic phlogistic reactions.
E. Marton, E. Giordan, P. Gallinaro et al. Journal of Clinical Neuroscience 89 (2021) 412–421

Fig. 1. Dura mimicking score. Pictorial representation of the degree of integrity (first row), adhesion to dura and thickness (second row), and AM adhesion to the cerebral
cortex (third row). The AM transparency reflects the degree of fibrosis and neoangiogenesis developed over time, while the AM thickness and dura mater progressive fading
into AM are reflective of differentiation, integrity, and integration to the dura, respectively. The ‘‘adhesion to the cerebral cortex” is an index to define how the AM patch tends
to adhere to the cerebral parenchyma. It highlights the malleability of the patch in cranioplasty reconstruction. From left to right: one point for the first column, two points for
the second, and three points for the third.

2.3. Amniotic membrane retrieval, processing, and storage saline, and samples of the rinsing solution were collected without
filtering for microbiological analyses. Microbiological analyses
AM donors were selected following the guidelines for were completed by an accredited in-hospital microbiology labora-
harvesting, processing, and distributing tissues for transplantation tory and interpreted by a microbiologist with specific expertise in
as approved by the National Transplant Center. Donor selection cri- this field.
teria include 1) donors without a medical history positive for The AM was cut into two different patch sizes: 12x10 cm or
malignancies; 2) no pediatric malformations or pathological condi- 15x12 cm. The processed AM is rapidly frozen at 50 °C to
tions detected in the prenatal period; 3) at least 35 weeks of gesta- 80 °C; then, it is vacuum-dried using a freeze-drying device.
tion; 4) familial history negative for genetic disease, and 5) both Water from the tissue is extracted through sublimation until a final
donor and partner without infectious disease risk behaviors. water content of 5–10% is attained.
Donor blood was screened for HIV-1 and 2 antibodies, HTLV-1 The lyophilized AM patch was stored in sterile envelopes. The
and 2 antibodies, hepatitis B surface antigen, hepatitis B core lyophilized AM patches were stored at 15 to 30 °C for no more than
antibody, hepatitis C virus (HCV), and syphilis. The screening pro- five years.
cedure also included testing IgM/IgG antibodies against toxoplas-
mosis and cytomegalovirus, as well as nucleic acid amplification
tests (NAT) for HIV, HBV, and HCV.
2.4. Description of decompressive craniectomy and cranioplasty
The AM was collected from the placenta of donors undergoing
techniques for AM patients
elective cesarean section. The procedure was performed in various
hospitals, which refer to our tissue bank (Fondazione Banca Dei
All patients underwent a standard wide hemispheric decom-
Tessuti di Treviso (FBTV)). All of the donors signed informed con-
pressive craniectomy. After bone flap removal, the dura was cut
sent before their delivery. Within 24 h and under sterile conditions,
into a stellate shape to allow brain expansion. The dry AM patches
the AM was carefully isolated from the rest of the placenta and
came inside a sterile envelope. A small paper marker was laid on
rinsed with saline solution to remove residual blood. Then, the
the chorion side to allow layer recognition. The AM patches were
AM stromal/mesenchymal layer was placed in contact with a nitro-
rehydrated in sterile saline solution for 10 min at ambient temper-
cellulose membrane filter (Merck Millipore), which acted as a sup-
ature to moisture the surfaces and were then mobilized with the
port for flattening the patch. At that point, the AM was covered
help of small surgical forceps. Each patch was then carefully lay-
with an antibiotic solution (vancomycin 100 lg/ml (Hospira)),
ered over the parenchyma and under the dural flaps. Although
meropenem 200 lg/ml (Fresenius Kabi Italia), and gentamicin
AM patches can easily be sewn to native dura flaps, we lay the
200 lg/ml (Fisiopharma) for 24 h at 4 °C.
AM patches under the dura mater flaps to favor brain expansion
Bacteriological examinations for aerobic and anaerobic bacteria
without impediments. Positioning AM patches is not different from
and fungi/yeast were performed upon processing and before
laying other kinds of dural substitutes that rarely require to be
freeze-drying the membrane. The AM was also rinsed with isotonic
sewn. No other dural substitutes were used after AM deployment.
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E. Marton, E. Giordan, P. Gallinaro et al. Journal of Clinical Neuroscience 89 (2021) 412–421

Fig. 2. A. Re-hydration of the lyophilized AM patch in sterile saline solution. B. Unfolding and flattening of the membrane.

(Fig. 2). We chose to lay the AM with the amnion side facedown
to favor this layer’s anti-adhesive proprieties on the brain parench-
yma. The chorion side was placed facing upwards, towards the
muscle flap and the dural folds’ inner face, to promote fusion and
integration (Fig. 3).
During the cranioplasty, the AM appearance was carefully
inspected by the surgeons and biopsied at multiple points at the
dural flap’s borders for pathological examination. The surgeon
reported parameters such as adhesivity to the dura mater or brain
parenchyma, defects in coverage, leakage of cerebrospinal fluid
(CSF fistula), and graft integrity. An experienced neuropathologist
then examined the AM specimens.

Fig. 3. A. Decompressive craniectomy: the dura is opened in a stellate fashion and the swollen parenchyma exposed. B. The AM patch is carefully placed, amnion side down. C.
The protective gauze, which is by default set on the chorion side, is gently removed. D. The AM patch is unfolded to cover all of the exposed brain adequately. E. Eventually,
the dural flaps are flipped over the AM. F. Right: bilateral decompressive craniectomy. Left: brain parenchyma covered by AM and dural layer.

415
E. Marton, E. Giordan, P. Gallinaro et al.
2.5. Histopathological processing of the AM samples
AM samples were retrieved during cranioplasty by sampling
multiple locations. A total of four samples were collected from
each AM layer at the time of cranioplasty. Two specimens were col-
lected over the cerebral parenchyma and the others near the native
dura mater. They were fixed in neutral buffered formalin (10%) and
embedded in paraffin wax and then cut into 4 mm thick-sections
and stained with hematoxylin and eosin (HeE). The immunohisto-
chemical analysis was performed using an automated immunos-
tainer, testing primary antibodies against CD3 (T-lymphocytes),
CD20 (B-lymphocytes), and CD68 (histiocytes).
2.6. Cranioplasty for biological dural substitute patients
During the cranioplasty, the dural substitute appearance was
carefully inspected by the surgeons, reporting parameters such as
adhesivity to the dura mater or brain parenchyma, defects in cov-
erage, leakage of cerebrospinal fluid (CSF fistula), and graft integ-
rity (i.e., compiling the ‘‘dura mimicking score”).
2.7. Statistical analysis
Descriptive statistics were reported as median and range for
continuous variables and number and percentage for categorical
variables. The Mann-Whitney U test was used to analyze the con-
tinuous variables and the Fisher exact test for the categorical vari-
ables. A p-value of < 0.05 was regarded as statistically significant.
All statistical tests were 2-tailed. All statistical analyses were per-
formed using Stata Version 13.0 (StataCorp, College Station, TX).
3. Results
3.1. AM cranioplasty patients
3.1.1. Patient demographics and characteristics
Twenty-seven patients were prospectively enrolled between
January 2015 and January 2018. Two patients died after decom-
pressive craniectomy due to refractory malignant intracranial
hypertension and thus were excluded from the final cohort. Our
final sample included 25 patients.
The median age at diagnosis was 51 years (range 26 – 68 years),
and the male to female ratio was 1.5 to 1, 40% of patients were
female. The majority of patients presented after an MCA / ICA
ischemic stroke (with consequent malignant ICH, 45%), followed
by subdural hematoma (35.0%) associated with malignant brain
edema, traumatic or spontaneous intraparenchymal bleeding
(15.0%), and epidural hematomas with concomitant multiple
intracranial contusions (5.0%).
There were no significant differences in the decompressive
craniectomy location (right vs. left, p-value: 0.530), and only two
patients underwent bilateral decompression. The GCS at operation
time ranged from 4 to 13, median 8. Median decompressive oper-
ative time was 98 min (range 50–159 min), while median blood
loss was 300 mL (range 50–600 mL). Thirty-five percent of patients
were using blood thinner therapy (antiaggregant or anticoagulant
drugs), and 45% had medically relevant comorbidities, but only
one was under immunosuppressive medications. The estimated/-
calculated median size of the dural defect needed to be covered
was 70 cm2 (range 60 – 110 cm2). Cranioplasty was performed in
a mean time of 61 days (range 36 to 88 days). After opening skin
and muscle flaps, the amniotic membrane was evaluated both with
macroscopic visual parameters (integrity, CSF fistula) and micro-
scopic findings after removing a small specimen (integration of
the AM with the connective tissue of the dura). Cranioplasty oper-
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Journal of Clinical Neuroscience 89 (2021) 412–421
ative time was 88 min (range 58 – 131 min) with a median blood
loss of 150 mL (range 50 – 300 mL). The median follow-up after
cranioplasty was 44.5 months (range 5 – 75 months).
3.1.2. Outcomes, complications, and macroscopic intraoperative
findings
Ninety percent of our patients were discharged from the inten-
sive care unit to the rehabilitation ward. We had one case with a
severe postoperative complication, a subacute subdural hema-
toma. The event occurred after the cranioplasty and was docu-
mented at follow-up CT scans. It occurred two weeks after the
reintroduction of antiaggregant therapy. The patient was re-
operated and fully recovered.
We detected a CSF collection in the subdural space at the
follow-up CT or MRI in three patients. Among those patients, one
developed CSF leakage and needed reintervention for the closure
of the fistula. Three patients also experienced severe postoperative
medical complications – all were nosocomial infections (two Kleb-
siella Pneumoniae pulmonary infections and one Pseudomonas
aeruginosa urinary infection). All patients were adequately treated
and fully recovered.
The majority (96%) of AM grafts were macroscopically well inte-
grated during cranioplasty, with apparent good fusion among the
AM patch and native dural flaps. In one case, we documented a
defect in AM integration to the dural plane, probably due to an
intraoperative malposition of the AM. In that case, the defect was
repaired with a fibrinogen matrix patch (TachosilÒ, Takeda Phar-
maceutical Company, Osaka, Japan).
According to our ‘‘dura mimicking score”; 13 patients scored
excellent, 11 good, and 1 poor.
3.1.3. Histopathological findings
In the majority of AM specimens (90%), histopathological find-
ings showed several thick plates of dense fibrous tissue character-
ized by numerous reactive small vessels, a predominant population
of reactive fibroblasts, and by a diffuse inflammatory infiltrate
made by CD3-positive T lymphocytes, CD68-positive histiocytes,
and scattered CD20-positive B lymphocytes (Fig. 4). Blood extrava-
sations and hemosiderin accumulations were present. Necrotic
areas were absent in all of the specimens. In one case, there was
a focal proliferation of meningothelial cells in vorticous nests. Only
one AM showed thin plates of fibrous tissue with no inflammatory
reaction. The amniotic epithelium was not evident in any of the
analyzed specimens. Histochemical stains (i.e., PAS and GMS),
showed neither fungi nor bacteria.
3.1.4. Postoperative imaging results
Contrast-enhanced brain MRI was performed in all patients
approximately one month after decompressive craniectomy.
Although it was not possible to quantify AM signal differences,
our inspection documented how the AM seemed consistently more
hyperintense at T1-CE pre-cranioplasty MRI sequences compared
to the immediate post-decompression ones. It seemed that the sig-
nal intensity was mimicking MRI alterations associated with the
neoangiogenesis process becoming gradually but linearly more
and more intense but never reaching the higher intensity signal
typical of inflammation and dural scar. Some illustrative cases
are reported in Fig. 5.
A posthoc sensitivity analysis stratifying AM conservation time
outcomes (<12 months vs. > 24 months) did not find any signifi-
cant differences in terms of the scores as mentioned above or
histopathological or imaging findings.
E. Marton, E. Giordan, P. Gallinaro et al. Journal of Clinical Neuroscience 89 (2021) 412–421

Fig. 4. AM histopathological characteristics: A. A thick fibrous plate rich in small vessels, moderate cellularity, and blood extravasation (HeE 100X) [BE: blood extravasation,
SV: small vessels]. B. Cellularity is predominantly made of reactive fibroblasts and monocitoid inflammatory cells (HeE 200X) [RF: reactive fibroblasts and spindle cells. MC:
monocitoid cells with black and round nucleus]. C. The proliferation of non-atypical meningothelial cells in vorticous nests (arrow) (HeE 100x) (inset 200X) [VN: vorticous
nests]. D. A thin fragment of fibrous tissue without a prominent inflammatory reaction (HeE 100X) [the line points at nervous tissue].

3.2. Biological dural substitute group 3.3. Outcomes, complications, and macroscopic intraoperative findings

3.2.1. Patient demographics and characteristics
Twenty-five patients were prospectively enrolled, and no
patients were excluded or lost at follow-up. The median age at
diagnosis was 45 years (range 20–59 years), and the male to female
ratio was 1.3 to 1; 44% of patients were female. Eighteen of the 25
patients had DURAFORMÒ in compressive craniotomy, and 7 had
TUTOPATCHÒ. The majority of patients had an ischemic stroke
(47%), followed by subdural hematoma associated with malignant
brain edema (28%), traumatic intraparenchymal bleeding (25.0%).
There were no significant differences in the decompressive
craniectomy location (right vs. left, p-value: 0.162). The GCS at
operation time ranged from 5 to 12, median 8. Median decompres-
sive operative time was 90 min (range 60–137 min), while median
blood loss was 225 mL (range 100–500 mL).
The dural defect’s estimated median size needed to be covered
was 75 cm2 (range 50 – 115 cm2). Cranioplasty was performed in a
mean time of 65 days (range 38–101 days) after craniectomy.
Twenty-five percent of patients were under blood thinner ther-
apy, and 20% had medically relevant comorbidities.). Cranioplasty
median operative time was 80 min (range 50–125 min) with a
median blood loss of 150 mL (range 50 – 300 mL). The median
follow-up after cranioplasty was 38 months (range 12–68 months)
(Table 1).
417
All of our patients were discharged from the intensive care unit
to the rehabilitation ward. In one case, postoperative CFS fistula
occurred, and in one other extradural blood collection was evident.
Those events occurred after the cranioplasty. The first patient was
re-operated, and he fully recovered. The second underwent close
imaging and clinical monitoring till spontaneous resolution of
the hematoma. Two patients experienced postoperative medical
complications – a Pneumonia infection and a Clostridium Difficile
contamination. All patients were adequately treated and fully
recovered.
According to our ‘‘dura mimicking score”; 7 patients scored
excellent, 13 good, and 5 poor.
4. Discussion
An ideal dural substitute must have: 1) the ability to act as a
structural and protective barrier for the cerebral cortex, maintain-
ing its integrity and preventing CSF leakage while not developing
excessive adhesions to the arachnoid/pia mater; 2) the ability to
provide a safe dissection plane during cranioplasty, preventing
excessive adhesions to temporal muscle and skin flaps. These pro-
prieties are linked to the maintenance of graft integrity and its
integration into native tissues [1,26–28].
E. Marton, E. Giordan, P. Gallinaro et al. Journal of Clinical Neuroscience 89 (2021) 412–421

Fig. 5. A. Pre-cranioplasty axial (left) and coronal (right) T1-CE contrast-enhanced MRI scans of a patient who underwent left decompressive craniectomy. Here we clearly
illustrate that the AM patch (white arrow) is more hyperintense and thick than the native dura on the right side (arrowhead). B. After-cranioplasty axial (left) and coronal
(right) T1-CE contrast-enhanced MRI scan of a patient in which an adhesive dural substitute patch was used. The dural substitute tend to not show any hyperintensity in
respect to the native dura (arrowheads). C. Again, pre-cranioplasty axial (left) and coronal (right) T1-CE MRI of another patient who underwent a decompressive craniectomy.
The AM patch is well integrated into the native dura and the thick hyperintense signal and increased thickness compared to native dura mater. D. MRI images of a patient
waiting for cranioplasty (axial (left) and coronal (right) T1-CE MRI). Again a dural substitute (a pericardium patch) was used instead of AM. The patch is not well integrated
(arrow), and not visibly more hyperintense than the native dura.

Table 1
Population baseline characteristics.

AM GROUP BIOLOGICAL GROUP

Age at decompressive craniectomy
(median ,range) [yr] 51 (26–68) 45 (20–59)
Female sex
N (%) 10 (40%) 11 (44%)
Cause of ICH N (%)
Ischemic stroke (MCA/ICA stroke) 11/25 (45%) 12/25 (47%)
Subdural acute hematoma + cerebral oedema (±cerebral contusion) 9/25 (35%) 7/25 (28%)
Intraparenchymal hematoma (spontaneous/traumatic) 4/25 (15%) 6/25 (25%)
Epidural hematoma + cerebral contusions 1/25 (5%) /
GCS pre decompressive craniectomy
(median, range) 8 (4–13) 8 (5–12)
Operative metrics: decompressive craniectomy
Surgical time (median, range) [min] 98 (50–159) 90 (60–137)
Blood loss (median; range) [mL] 300 (50–600) 225 (100–500)
Operative metrics: cranioplasty
Surgical time (median, range) [min] 88 (58–131) 80 (50–125)
Blood loss (median; range) [mL] 150 (50–300) 200 (50–300)
Relevant therapy
Blood thinner N (%) 9/25 (35%) 6/25 (25%)
Immunosuppressive N (%) 1/25 (5%) /
Dural defect area – estimated size
(median, range) [cm2] 70 (60–110) 75 (55–115)
Follow-up
(median, range) [months] 44.5 (5–75) 38 (12–68)

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E. Marton, E. Giordan, P. Gallinaro et al.
Four types of dural substitutes are commonly used in
decompressive craniectomies: autograft, allograft, xenografts, and
synthetic grafts [29,30]. Theoretically, autografts such as fascia lata
or galea/pericranium are the ideal solution because they seem not
to invoke an inflammatory response [31]. However, harvesting a
large graft (i.e., big enough to cover craniectomy defects), is diffi-
cult and may increase surgical morbidity [32,33]. Therefore, it is
more common to use biological or synthetic dural substitutes.
Recent literature reveals that synthetic and biological grafts reduce
operative blood loss and time during the subsequent cranioplasty
[26].
To date, there is a wide range of different organic or synthetic
dural substitutes, and the surgeon must choose based on personal
preference, availability, and cost. Unfortunately, the literature data
do not provide consensus regarding the types of complications
associated with each material [26,32]. Moreover, complications
have most often been evaluated in control groups, limiting the gen-
eralizability to clinical settings [26]. Furthermore, the phenomenon
of graft encapsulation and neo-membrane formation has not been
addressed in any previously published studies. Although previous
studies have compared the outcomes in terms of layers reconstruc-
tion during cranioplasty [26,27,32,34,35], dural substitutes’ anti-
adhesive properties have not yet been examined. AM is also
slightly immunogenic [12,14,16] and contains growth factors that
promote cell differentiation and healing [13]. Simultaneously, with
its ‘‘slippery” epithelium, the amnion has the potential to promote
an anti-adhesive effect [12].
To rule out AM characteristics, we randomly collected two
homogeneous groups of patients. We found no statistical differ-
ences in mean age and sex distribution (p-value: 0.206 and
0.776, respectively), underlying pathology distribution (p-value:
0.888), mean dural defect area to cover (p.-value: 1.000), mean
intraoperative time for decompressive (p-value:0.116) and adverse
events (both postoperative surgical and medical, p-value: 0.556),
and meantime from decompressive to cranioplasty (p-value:
0.206) between the two groups of patients. Slight differences were
found in the cranioplasty procedure time (p-value: 0.090) and
mean blood loss in cranioplasty (p-value: 0.172), that do not reach
statistically significant values, probably due to the small samples of
the two groups.
At our Institution, we started using AM ten years ago to take
advantage of its insulating properties, including watertight dural
closure and CSF leakage prevention [3]. Some evidence suggests
that AM prevents fibroblast migration into the exposed dura dur-
ing the early healing process; this is potentially the most crucial
aspect for reducing fibrosis [18]. Furthermore, the interposition
of a physical barrier to stem cell migration is considered another
effective strategy to minimize adhesions [19]. Our ‘‘dura mimick-
ing score”, although specifically designed for this study and not
externally validated, was, in our opinion, representative of the
integration capabilities of different dural substitutes. It highlights
how synthetic/biological substitutes tend to have less integrative
abilities (‘‘dura mimicking score” score poor: 20% vs. 4% for syn-
thetic/biological substitutes and AM respectively, p-value 0.085).
In our experience, AM patches formed a uniform layer with dura,
well-demarcated from the cerebral cortex, in almost all the
patients, thus serving as an excellent scaffold for subsequent
reconstructive surgery during cranioplasties. The AM group out-
comes were in line with those of the biological dural substitute
one.
The microscopical analysis of the specimens revealed the per-
fect integration of the amniotic membrane to the autologous dura,
along with epithelium’s disappearance and the development of
new connective tissue with thin plates of fibrous tissue with no
419
Journal of Clinical Neuroscience 89 (2021) 412–421
inflammatory reaction or necrosis. These results could be likely
associated with the anti-adhesive and pluripotential
differentiation properties of AM.
Additionally, the slight AM hyperintensity documented in MRI
has to be taken into consideration. The radiological evolution sug-
gests that AM does not behave like a simple barrier but adapts to
the environment and gradually develops into new vital tissue. That
correlates with histopathological findings, like the amnion’s disap-
pearance and its substitution by a delicate connective tissue. The
absence of high hyperintensity in contrast-enhanced MRIs may
reflect the lack of scar tissue, correlating once again with the
pathological findings. However, the MRI findings’ reasoning is
purely speculative and based on our experience, but we believe it
reflects the AM patches’ peculiar proprieties.
Our cranioplasty operative time and blood loss values were
within the lower ranges reported in the literature (range 61 –
121 min for operative time and range 110 – 220 mL for operative
blood loss) [36,37] and overlapping those of the synthetic/biologi-
cal group. Also, disease transmission risk is virtually impossible
because of the specimen’s careful retrieval and storage, along with
the competition of a full panel of microbiological tests on each
specimen.
We do not have enough power to evaluate the risk of epilepsy or
hydrocephalus development after AM positioning, but they will be
important outcomes to assess in future research. It should be noted
that, in our sample, a patient with a subdural hematoma had sei-
zures both in the pre-cranioplasty period and in the post-
cranioplasty period. However, it is impossible to know whether
the seizures resulted from irritation caused by hemoglobin or sub-
sequent AM cortical-arachnoid/pial adhesion. Future research
should evaluate seizures in patients, without previous hemorrhage,
with different adhesive dural-cortical patterns.
Eventually, we believe this is a viable option to consider in the
future. Although we cannot show the AM patch’s superiority com-
pared to the available options, AM offers unique proprieties that
need to be further evaluated in the future.
5. Limitations
This study has multiple strengths, including the novelty of the
topic. This is the first study that systematically considers integra-
tion and adhesion proprieties for a dural substitute in cranioplasty
to the best of our knowledge. The study also focused on the stan-
dardization of the procedure for processing AM. However, various
limitations must also be considered. The first limitation of this
study is the small sample size, limiting our ability to make prog-
nostic considerations. Furthermore, our follow-up time may not
have been sufficient to define the AM patch’s safety, especially in
terms of biological safety and postoperative seizure development.
Future studies with longer follow-up time are therefore needed.
We are aware that the lyophilized AM membrane is associated
with high retrieval, processing, and delivery costs. Overall, we esti-
mated that an AM patch of 15x12 cm is 60 to 90% higher than the
price of other common synthetic (i.e., collagen-based biocompati-
ble material) or biocompatible (i.e., xenografts, bovine peri-
cardium) dural substitutes of the same size. Also, the cost of a
large patch of dural substitutes (i.e., 12x10cm DURAFORMÒ patch
is 1.400$) is not so different from the price of processing an AM
patch (approximately 2.800$), and revision surgery costs need to
be considered for in the final analysis. Thus, AM patches are not
cheaper than other dural substitutes. However, in our Institution,
we have the advantage of cooperating with an internal tissue bank
that is efficient and prompt in perceiving needs and furnishing AM
E. Marton, E. Giordan, P. Gallinaro et al.
patches for operative use. Thus, we got used to AM disponibility
and had the opportunity to manage tissue defects in different fields
of neurosurgery and test its superb qualities.
Also, for lyophilized AMs, the storage costs are virtually nonex-
istent, considering that the lyophilized AM patches can be stored
for several years at ambient temperature. Eventually, although
simple, our dura mimicking score is an effective way to assess
AM patch behavior. The score is directly dependent upon the sur-
geon’s opinion, which may reduce the score’s objectiveness, but
the way it is designed is easily reproducible.
6. Conclusions
Decompressive craniectomy is a common neurosurgical proce-
dure, often using synthetic or biologic grafts as dural substitutes.
However, no consensus has yet been reached on the best dural sub-
stitute to use. In this study, we analyzed two of the most important
factors for decision making – antiadhesive properties and integra-
tion to native dura mater abilities– when using human lyophilized
AM. Our sample highlighted how the AM meets both of these cri-
teria and reduces risk of morbidity and infection in patients, and
it is not inferior to other dural substitutes. Eventually, our work
could be the pilot project for a multicenter study to evaluate
AM’s efficacy and safety as a dural substitute in emergent and elec-
tive surgery.
Sources of funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Ethical approval
All procedures performed in studies involving human partici-
pants were in accordance with the ethical standards of the institu-
tional and/or national research committee and with the 1964
Helsinki declaration and its later amendments or comparable eth-
ical standards.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgment
None.
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