Dry Human and Bovine

Biological Dressing

Use of
as a
T. Viswanatha

Amnion

Rao, MS, V. Chandrasekharam, MS

\s=b\ A new method of drying and sterilizing the amnion extends

the life of the product to more than nine months and enables it to
be stored at room temperature. The bovine amnion is unique and
the first of its kind, there being no comparable series in
the literature, to our knowledge. Bovine amnion has a large surface
area and can provide bulk supplies. The membrane as prepared
has been tested in 12 rabbits experimentally and in 70 patients of
different ages with a variety of surgical lesions, mainly burns. More
than 150 applications of the dried amnion have been made as the
sole dressing for these patients. The results indicate that amnion,
both human and bovine, is quite safe and effective as a biological
dressing. The dried material retains all the physical and biological
characteristics of the fresh membrane.

(Arch Surg 1981; 116:891-896)

idea of fetal membranes as a substitute for skin
was first suggested by Davis.1 The first clinical trial is
to Sabella.2 Soon it was realized that these mem
branes were useful as temporary biological dressings only.
Johnson3 suggested the use of bovine allantois as a source
of biological dressing. Chao et al4 were the first to use the
dried and alcohol-preserved amniotic membrane for craniotomy wounds to cover extensive durai defects and to
prevent adhesions. They observed severe reactions when
chorion was retained for a short time before application.
Troensgaard-Hansen5 used amniotic grafts for the treat
ment of chronic nonhealing ulcers. Allografts of skin from
donors and cadavers were popularized by Brown et al.6
Silvetti et al7 suggested the use of xenografts from bovine
embryo skin.
Interest in fetal membranes has been revived recently,

The
credited

Accepted for publication Oct 7, 1980.

From the Department of Pediatric Surgery, Andhra Medical College,

Visakhapatnam, India.
Reprint requests to Department of Pediatric Surgery, Andhra
College, Visakhapatnam 530 002, India (Dr Rao).

Medical

with multiple reports having been published in various

journals. Human amniotic membrane has been used in all

these studies. The main drawback of these membranes

appears to be the difficulty of assuring adequate supplies
and the difficulty of their preservation. Dino et al8 studied
the effect of human amniotic membranes in burns. They
described the method of processing the membrane and
suggested the organization of an amnion bank.
The earlier attempts at preservation of amnion de
scribed by Chao et al seem to have been abandoned, as no
further reports are available of their methods having been
pursued. Dino et al have preserved the amnion at 4 C after
repeated rinses in saline solution. When kept in saline
solution at room temperature, 100% contamination of the
specimens occurred within 48 hours. Addition of 0.25%
sodium hypochlorite to the saline solution kept specimens
free of infection for one month. Addition of 200,000 units
of crystalline penicillin improved the viability up to six
weeks, especially if stored at 4 C. This method is very

popular

and is

extensively

used

by

most workers in the

field.
We have attempted a simpler technique that eliminates
the need for a costly amnion bank and that enables storage
at room temperature. The twin problems of preservation
by a simple technique and provision of adequate supplies
when most needed will be solved by (1) preserving the
amnion in the dry state and (2) using the bovine amnion to
provide bulk supplies. This communication recounts our
experiences with these two products.
Table 1 gives the characteristics of the two types of
amnion. The large surface of the bovine amnion is worth
noting. Our figure for the human amnion is at variance
with that of Troensgaard-Hansen,5 who claims only 52 sq
cm (8 sq in) of amnion as being available from a single

placenta.

The normal

requirement

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of

biological dressing

for 50%

Fig 1 .Bovine amniotic sac reconstituted by filling with tap water.

Note large surface of sac. It had to be filled with 15 L of tap water

to distend it. It has been folded for convenience.

Fig

burns in an adult has been estimated to be about 6,000 sq

cm.9 Such large quantities of human amnion will be
difficult to procure, especially if the amnion has to be

2.Dried amnion sealed in

not available in many

parts of the world, whereas

amnion, both human and bovine, is available universally at

almost

no

cost.

SUBJECTS AND METHODS

Fresh placenta is procured at normal, healthy deliveries from
without a history of premature rupture of membranes. The
amnion is separated from the chorion and the fetal surface of the
placenta up to a short distance from the implantation of the cord,
care being taken not to include Wharton's jelly as it prevents
proper drying. The membrane is washed in saline to remove clots
and debris. Finally, it is rinsed in saline solution containing a little
bleaching powder. The membrane becomes bleached as well as
sterilized by this method. It is now spread on a plastic sheet and
allowed to dry in the open air. It dries within a short time and
adheres to the plastic sheet. The membrane can easily be sepa
rated from the plastic sheet. It is then sealed in polythene covers
and labeled. The method of processing and drying the bovine
amnion is identical (Fig 1).
The dried and sealed covers (Fig 2) containing amnion are
sterilized by (1) ultraviolet (UV) rays or (2) y rays. For routine use,
UV sterilization was quite adequate. One hundred packets of
amnion were sterilized by y radiation. Random samples of amnion
were tested bacteriologically for sterility.
women

Experimental

Animals

Twelve rabbits weighing 900 to 1,400 g were given a uniform

deep burn of 7.6 x 2.5 cm (3 X 1 in) under ether anesthesia. Five

days later, escharectomy was followed by the application of

amnion (bovine only) to half of the wound, the other half being left
open as a control. The amnion was not sutured to the wound edges;
it was simply allowed to adhere to the wound surface. Repeated
inspections of the wound were made at intervals of five days each

covers.

until the healing was complete. Cultures were taken from the
wound surfaces during these inspections. No other treatment was
given to the animals.

changed every 48 hours. Allografts and xenografts of skin

are

polythene

Clinical

Subjects

Seventy patients with various surgical lesions, 58 of them from

burns, are the subjects of this study. These 70 patients had more
than 150 applications of the amnion. The detailed particulars of
the patients are given in Tables 2 through 4.
Superficial Burns.The wound was prepared by excision of blebs
and slough. It was washed with sterile saline solution. Amnion,
human or bovine, was applied to the raw surface and allowed to
dry. Except in the first ten patients, only the dried and sterilized
amnion was applied to wounds. No sutures or retaining dressings
were applied. Local antibiotic creams were avoided. Antibiotics for
a systemic effect and intravenous fluids were administered as
needed.
Deep Burns.The routine just described was modified slightly.
Immediately after admission, as much of the area as possible was
covered with amnion without making any effort to assess the
depth of the wound. Blebs and overhanging skin and obviously
dead tissue were excised. These areas also were later covered with
amnion. Escharectomy was performed at the earliest date after
renal function was reestablished, usually around the fifth day. The
raw area left after escharectomy was covered with amnion. In
extensive burns, escharectomy was performed in stages. Except in
cases with gross local sepsis, antiseptic lotions were not used
routinely. The amnion was changed only when signs of infection
were present or when the membrane showed signs of autolysis.
Pressure Sores.Because of gross infection in these cases, the
wound was thoroughly cleansed with antiseptic lotions using
povidone-iodine (Betadine), and slough was excised prior to appli
cations of the amnion. In deep excavating ulcers, amnion was also
packed into the depths of the ulcer. The dressing was changed
repeatedly at least once daily.
Ruptured Meningoceles.The site of CSF leak was identified and
the area was covered with amnion after the wound was cleaned. In
case of persistence of the leak or in postoperative cases with

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Table 1 .Characteristics of Amnion

Bovine

Human

Surface area,"

1,600

6,000-7,500

Shape

Spherical

Cylindrical

Thickness, /im

10

Histologie ap

Single layer

sq

cm

15

Single layer of

of

cuboidal

cuboldal

pearance

epithelium

'Surface area is calculated

Table

as

circumference

epithelium
length or height.

of Cases

2.Types

Amnion

Human
Fresh
Humans
Burns
Skin graft donor sites

Dry

Bovine,
Dry Only

Total

15

32

58

20

39
12

70
12

Ruptured meningoceles
Pressure
Total
Rabbits

3Burns induced in rabbit are covered with bovine amnion.

Membrane is still adherent on fifth day. Membrane-covered area,
which is clean and transparent, can be recognized easily. Control
area is opaque and covered by eschar. Note that no sutures hold
amnion.

Fig

sores

11

Table 3.Patient Characteristics: Age and Sex

Sex
Total
16
19

Age
11

0-1 yr
1-4 yr
5-8 yr
9-12 yr
Adult
Total

15
11
23

47

70

The male-female ratio is 2:1.

Table 4.Surface Area and

Area, %

Degree

Mixed

Deep

Superficial

of Burns

23
10
10

16

10
20

30
40
50
>50
Total
Data for the

wound

50s

20

29

remaining eight

sepsis only,

Total

cases are

the amnion

was

missing.

changed after

24 to 48

hours.

RESULTS

Experimental Animals
The safety and effectiveness of the bovine amnion has
been established. All of the rabbits survived the procedure.
The wounds healed within three weeks. The membrane
adhered to the wound surface and remained adherent
throughout the period of healing, even though it was not
sutured and a dressing was not applied. A single applica
tion was enough.

Fig 4.Baby
man).

with

superficial burns dressed with

amnion

(hu

The objectives of our study on the rabbits were limited to

the evaluation of the safety, durability, and bacteriostatic
activity of the membrane and the effectiveness and adher
ence of the bovine amnion. In all these respects, the bovine
amnion has proved satisfactory. Cultures from the amnion
covering half of the wound were sterile while the control
area showed coagulase-positive Staphylococcus or Pseudomonas pyocyanea organisms. The amnion-covered area
remained transparent and clean, while the control area was
opaque, dirty, and covered by dense eschar (Fig 3).

Subjects
A single application was adequate in superficial burns,
except in sodden areas like the perineum. The membrane
Clinical

adhered to the wound and remained adherent until the

regeneration was complete. In the case of deep burns, the
amnion adhered both to the eschar and to the raw surface
following escharectomy. However, in view of the fullthickness loss of skin and due to the frequent occurrence of

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5.Child with extensive superficial

burns treated with bovine amnion. On the
fifth day, amnion is dried and adheres well
to wounds. In areas it peels off as healing
takes place from periphery.

Fig

Fig 6.Boy with

burns due to hot oil. Two

months after accident, wounds were
treated with human amnion. Except for
small area in front, rest of wound has
healed without need for grafting.

infection in these cases, the amnion had to be changed once

in 48 hours. In one case, the amnion had to be changed ten
times in about eight weeks (Fig 4 through 6). All the
superficial burns have healed without incident. None of
them required skin grafting. We note that the area
requiring skin grafting has also been reduced in the case of
deep burns. As healing occurred from the periphery, only
patchy islands in the center required grafting. Contrac
tures and deformities are not eliminated by the use of
amnion, hence some form of splints is needed to prevent
them.
There were no differences between the various types of
amnion used. This indicates that the process of drying has
not affected the physical or biological properties of the
amnion. Bovine amnion, when applied to either humans or
the experimental animals, behaved like an allograft, and
this behavior was similar in the human amnion. This
indicates that the bovine amnion is a safe and useful
biological dressing that is similar and perhaps superior to
heterografts of skin. Apart from its large size, bovine
amnion was more smooth and glistening, but it crumpled
more easily and got displaced from the wound more often,
indicating less efficient adherence to the wound.
There were no ill effects from the use of irradiated
amnion. Irradiation did not appear to have affected the
physical or biological properties of the amnion, either.
The results were further assessed as follows:
1. With respect to patient acceptance (as indicated by
relief of pain), patient comfort, and the absence of reac
tions (local or systemic), all three types of amnion have
given equally good results.
2. With respect to prevention of fluid losses, amnion
acted as an excellent vapor barrier by preventing further
losses of fluid from the wound bed because it adhered to
the wound surface firmly.
3. Patient morale was good in all cases.
4. The intimate and close bond between the wound and
the amnion effectively prevented infection from the exte-

rior. In full-thickness burns, infection was already present

within the wound. In these cases, repeated applications of
the membrane after early escharectomy controlled the
infection to a great extent.
The results in full-thickness burns were not uniformly
encouraging. However, surgical debridement and amnion
cover over the area reduced the pain and shock. The
patients became more comfortable after the application of
amnion, thus improving patient morale. Infection could
also be reduced by the method.
The period of convalescence was not significantly modi
fied by the use of amnion. But since the patients were more
comfortable and mobile, some of them could be treated as

outpatients.

Amnion was equally useful in nonthermal injury. In

ruptured meningoceles, amnion sealed off the wound and
prevented the inevitable CSF leak. In pressure sores,

infection was controlled in a short time and small ulcers

healed by themselves. Larger ulcers were covered by
healthy granulations flush with the skin so that skin
grafting could be undertaken.
After the wounds healed in superficial burns, the amnion
peeled off from the surface of the wound. However, cases
with full-thickness losses showed healthy pink granula
tions that remained flush with the skin and to which the
amnion remained adherent. The granulations formed were
never exuberant. Changes of dressing did not cause either
discomfort or bleeding, which are common with other
forms of dressing.

COMMENT

Terminology

and

Types

of 'Amnion'

"Amnion" and "amniotic membrane"

are

terms used

loosely in literature. Most authorities in this field have

used "amnion" mainly. Colocho et al10 removed the chorion
before processing the membrane and still called it "amniot
ic membrane." Walker et al11 separated the chorion from
the amnion and called the final product "amnion mem-

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brane." Chao et al' removed the chorion, dried the remain

ing membrane, and called it "amnioplastin," which they
preserved in alcohol. Troensgaard-Hansen5 also removed
the chorion and called the final product "amniotic graft."
Jenner,12 and later Dino et al," studied the effects of the
two membranes separately and concluded that there was
no difference between the various layers of the amniotic
membrane. Douglas et al1' noted vascular penetration
when chorion was used. Trelford et al14 did not find any
neovascularization when amnion alone was used. They
have actually suggested that chorion may be antigenic and
may be rejected immunologically. Walker et al11 did not
find any immunological reactions from the amnion pre
pared by them. The role of chorion, according to them, is
still undefined. Troensgaard-Hansen5 has used the smooth
surface of the amniotic graft to face the wound surface, as
he thought that the active principles of the membrane were
present only in the amnion.
The amnion prepared by us is similar to that prepared
by most of the authors just mentioned, except that it is
in the dried state. There have been no local reactions,
immunological or otherwise. There were no systemic
reactions either. The membrane retained all the physical
properties of the fresh amnion, along with its bene
ficial effects on the patient. We have observed, moreover,
that in places where the chorion was retained, the mem
brane did not dry properly and the dried product
remained sticky and waxy. It was also denser and
opaque.
We find in the literature that Robson and Krizek15 did
not make any distinction between the various layers of the
fetal membranes. They have used the complete amniotic

membrane.
Amnion seems to satisfy all the criteria of an ideal
biological dressing: an effective vapor barrier, adherence,
durability, bacteriostatic activity, protection from pain,
and elasticity.16

Vapor Barrier
Amnion is an excellent vapor barrier. It resembles in this

respect the stratum corneum epidermidis of the skin, which

is the natural vapor barrier,17 yet it has only one third the
thickness of the stratum corneum epidermidis (10 to 15 um
vs 30 to 60 um).
It is important to note that amnion does not influence
the shift of fluid in the extracellular space from the plasma
into the interstitial compartment. However, by preventing
evaporation from the wound surface, insensible losses,

which may exceed 2 to 3 L in burns greater than 18% to

40%,17 are prevented, thus reducing the quantity of fluid
required. Furthermore, by preventing evaporation from
the wound surface, the temperature regulation mechanism
is not overstrained and the caloric requirement needed to
maintain the temperature is also correspondingly reduced.
For this to be really effective, amnion should be applied at
the earliest opportunity from the time of burning. For
precise evaluation of the role of amnion in reducing the
hypermetabolism and fluid and electrolyte balance, further
studies are being made.

Physiological

and

Biological Properties

Amnion is a physiological-biological dressing since it is

derived from the epiblast and is continuous with the
surface ectoderm of the fetus. It is, as Pigeon18 has
remarked, an "extension of the baby's skin" and hence is
the most physiological of the biological membranes. Silvetti et al7 have advocated the use of bovine embryo skin
grafts. Structurally, amnion is made up of cuboidal epithe
lium on a framework of mesenchyme. The epithelium,
which is derived from the epiblast and which appears
before the embryonic plate is formed, remains in its
primitive state and fails to differentiate further. This
primitive state, or less complete differentiation of the
amniotic membranes, is considered to be the cause for their
antibacterial activity.1' Immunological reactions are also
considered to be absent because of this.
The bacteriostatic activity of the amnion is said to be due
to the presence of antibodies, possibly allantoin and lysozyme.15 It is not affected by the process of drying or by the
removal of the chorion, as experience shows. This indicates
that the dried amnion retained all the antibodies in a
dormant state and that they were not destroyed by the
process of drying, irradiation, or storage. The active prin
ciples present in the amnion appeared to be stable under all
these conditions. The dry membrane adhered as well as the
fresh one and remained adherent until wound regeneration
was complete, when it peeled off from the surface of the
wound by itself. In our experience, the bovine amnion has
behaved as well as the human amnion in the dry state.
Douglas et al" have noted very little difference between
heterologous and homologous chorion grafts in the mouse.
Our experience with the bovine amnion both in the rabbit
and the human subject tends to support this view. The
main advantage of the bovine amnion is its large surface as
compared with the human material. This availability of
large surface overshadows the minor disadvantage of less
efficient adherence to the wound. We have found no
comparable series in literature in which the bovine amnion
was used as the sole biological dressing.
Irradiation
Irradiation has been quite effective and safe in steriliz
ing the amnion. There were no deleterious effects of
radiation either on the amnion or on the host tissues.
Ultraviolet ray sterilization may be adequate for routine
external use of the membrane, but it fails to eliminate
spore-bearing organisms, as 70% of our samples were
reported to contain the nonpathogenic aerobic spore-bear
ing organisms. To eliminate these also, y radiation was
tried, and we found that the standard doses of y radiation
(2.5 mrad) routinely administered for all materials is quite
safe and effective for amnion also.

Bonding

Amnion, when spread on a plastic sheet, adheres to it as

it dries. But when it is separated from the wound surface
by interposition of a plastic sheet between the membrane
and the wound bed, it becomes ineffective. Thus, the

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behavior of amnion differs in this respect from a polyethyl

ene film. The bond between the two is similar to and more
than that of homografts and heterografts of skin rather
than a polyethylene film. There seems to be a biological,
not mechanical, bond between the wound and the amnion.
This biological bond has been considered to be a fibrin
elastin biological bond.19

Advantages

especially useful in children because the

membrane, being wafer thin and light in weight, releases
the children from the restraining influence of bulky dress
ings, which may cause respiratory embarrassment when
applied over the trunk and abdomen.
Use of dried amnion, it is hoped, will enable efficient
management of burns even in rural surroundings. The
importance of the dry membrane will be appreciated
Amnion is

developing countries like India, where main

costly amnion banks is not easy except in major
cities. Furthermore, bank amnion has a life span of only six
weeks, whereas the dried membrane has a life span of at

especially

in

tenance of

least nine months in our hands. This method of treatment

does not strain the finances of the patient or the hospital
services. The use of bovine amnion provides unlimited
supplies of the material. The effectiveness of dried and
irradiated amnion also confirms that tissue viability is not
essential.
K. L. Dutt, GMVC, ND(AR), PG (AUS), joint director and project officer
of the Indo-Swiss Cattle Research Station, Visakhapatnam, India, provided
the bovine amnion; J. Seethamma, MD, DGO, superintendent of Victoria
Hospital for Women and Children, Visakhapatnam, provided the human
amnion; and P. A. Jayakar, MD, DCh, professor of microbiology, and B. Raja
Rao, MD, additional professor of microbiology, Andhar Medical College,
Visakhapatnam, provided the experimental animals.

References
1. Davis JS: Skin transplantation, with a review of 550 cases at the John
Hopkins Hospital. Johns Hopkins Hosp Rep 1910;15:307.
2. Sabella N: Use of foetal membranes in skin grafting. Med Rec NY

1913;83:478.

3. Johnson HL: Insulating patches and absorbable sutures made from

fetal membranes. N Engl J Med 1937;216:978-982.
4. Chao YC, Humphrey SS, Penfield WA: A new method of preventing
adhesions: The use of amnioplastin in craniotomy wounds. Br Med J

1940;1:517-519.

5. Troensgaard-Hansen E: Amniotic grafts in chronic skin ulceration.

Lancet 1950;1:859-860.
6. Brown JB, Fryer MP, Rendall P, et al: Post mortem homografts as
biological dressings for extensive burns and denuded areas. Ann Surg

1953;138:618-630.
7. Silvetti AN, Cotton C, Byrne RJ, et al: Preliminary studies of bovine
embryo skin grafts. Transplant Bull 1957;4:25.
8. Dino BR, Eufemio GG, DeVilla MS: Human amnion: The establishment
of an amnion bank and its practical applications in surgery. J Philip Med
Assoc 1966;42:357-366.
9. Song IC, Bromberg BE, Mohn MP, et al: Heterografts as biological
dressings for large wounds. Surgery 1966;59:576.
10. Colocho G, Graham WP III, Greene AE, et al: Human amniotic

membrane as a physiologic wound dressing. Arch Surg 1974;109:370-373.

11. Walker A, Cooney DR, Allen JE: Use of fresh amnion as a burn
dressing. J Pediatr Surg 1977;12:391-395.
12. Jenner JA: The Grafting of Preserved Fetal Membranes to Denuded
Skin Surfaces. Marquette University, Milwaukee.
13. Douglas B, Conway H, Stark RB, et al: The fate of homologous and
heterologous chorionic transplants as observed by transparent tissue chamber technique in the mouse. Plast Reconstr Surg 1954;13:125-129.
14. Trelford JD, Anderson DG, Hanson FW: Considerations of the
amnion as an autograft and as an allograft in sheep. J Med 1972;3:231.
15. Robson MC, Krizek TJ: The effect of human amniotic membranes on
the bacterial populations of infected rat burns. Ann Surg 1973;177:144\x=req-\
149.
16. Shuck JM: The use of homografts in burn therapy. Surg Clin North
Am 1970;50:1325-1335.
17. Gump FE, Kinney JM: Caloric and fluid losses through the burn
wound. Surg Clin North Am 1970;50:1235-1248.
18. Pigeon J: Treatment of second-degree burns with amniotic membranes. Can Med Assoc J 1960;83:844.
19. Burleson R, Eiseman B: Nature of the bond between partial-thickness
skin and wound granulations. Surgery 1972;72:315-322.

Invited Editorial Comment

This report by Drs Rao and Chandrasekharam describes the use
of dried amnion from both human and bovine sources as a
biological dressing. Their examples of multiple kinds of wounds,
both experimental and clinical, are quite impressive. They have
found that amnion has most of the properties of a biological
dressing. However, one function that it does not fulfill is that of a
biological-physiological dressing. Because the chorion has been
stripped from the amnion, it cannot have vascular ingrowth.
Studies from our laboratory and others have shown that the
effectiveness of decreasing tissue bacterial counts is due to the
vascular ingrowth into the biological-physiological membrane.
Finally, there is difficulty in using any type of biological
dressing over a full-thickness burn. A full-thickness burn by
definition will have nonviable tissue and bacteria in its depth. To
close this with a biological dressing is very much like putting a top

abscess. The authors had difficulty using it over a fullthickness wound, and indeed this is the experience of others who
have tried either a biological dressing or a synthetic dressing such
as Hydron Wound Dressing (Abbott Laboratories, North Chicago,
111) over full-thickness injuries. Biological dressings are best used
for partial-thickness injuries or full-thickness injuries after the
eschar has been totally excised.
Despite objections to the specific points mentioned, the article
describes a way to dry amnion and preserve it for long periods of
time. If one is desirous of a biological dressing to serve only as a
cover and not function as a biological-physiological dressing, these
techniques may prove to have value.
Martin C. Robson, MD
on an

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Chicago