,3
`
`
`
`3mm. JournalafPlarrir singly (1939). 42463-461
`© I989 The Trustees of British Association of Plastic Surgeons
`
`0007~l226l89l0042—0463/510.00
`
`The healing of chronic venous leg ulcers with prepared
`human amnion
`
`D. J. WARD, J. P. BENNETT. H. BURGOS and J. FABRE
`
`Queen Victoria Hospital, East Grinsread
`
`Summary—Forty chronic venous leg ulcers were treated, before split skin grafting, with human
`amnion prepared in one of the four following ways: tissue-culture-maintained, frozen. fresh or
`lyophilised. Although there was no significant statistical difference in the results obtained with the
`different preparations of amnion. we found that lyophilised amnion was at least as good as the other
`preparations in promoting a good take of the skin grafts and was the simplest to store and use. it also
`produced the shortest healing times. Frozen and fresh amnion were easier to prepare than lyophilised
`amnion but gave a lower graft take and a longer healing time. Tissue—culture-maintainad amnion was
`the most difficult to prepare and gave the poorest results. Its use was abandoned during the trial
`because of technical difficulties and a high infection rate.
`
`Human amnion has previously been shown to
`stimulate both granulation tissue and new vessel
`formation in chronic venous leg ulcers (Faulk et al.,
`1980). When applied to the ulcers prior to skin
`grafting, amnion promoted a good take ofthe grafts
`(Bennett et (11., 1980). The angiogenic and growth-
`promoting factors which are present in amnion
`have now been identified (Burgos, 1986).
`These previous studies used amnion maintained
`by tissue culture. This is a complicated technique
`requiring laboratory facilities and regular super-
`vision and is not practical for everyday use. Lyo-
`philised, frozen and fresh amnion have been used
`in the past to promote healing in a variety of
`wounds (Matthews et al., 1982): frozen and freshly
`prepared amnion gave good results (Sabella, 1913;
`Sterling, 1956; Dino er 01., 1966) but lyophilised
`amnion had a variable degree of success (Notea er
`a1., 1975; Unger and Roberts, 1976).
`We designed a trial to compare these three other
`preparations of amnion with tissue-culture—main-
`tained amnion,
`looking at ease of preparation,
`storage and use, and each one’s capacity to promote
`wound healing. Chronic venous leg ulcers were
`treated with fresh, lyophilised, frozen and tissue-
`culture-maintained amnion before split skin graft-
`ing and the results were compared.
`
`Materials and methods
`
`within 2 weeks of term. Patients with any condition
`predisposing to placental
`insufiiciency such as
`diabetes were excluded, as were patients with
`meconium staining of the liquor. A sample of cord
`blood was taken and the serum was tested for
`Hepatitis B. This was found to be negative in all
`cases. The blood was not tested for HIV as this trial
`was performed before HIV became widespread.
`The amnion and chorion were detached from the
`placenta under sterile conditions in the delivery
`Suite and were transported to the laboratory in ice-
`cold phosphate-buffered saline (PBS)
`(pH 7.4)
`where the chorion was separated from the amnion
`and discarded.
`
`After cleaning and rinsing in PBS the amnion
`was divided into four pieces and each quarter was
`processed in one of the following ways:
`
`1 . Tissue—culture-mainzained
`
`5x 5 cm squares of amnion were maintained in
`tissue culture at 37°C for a maximum of 3 weeks
`using a modification of the method of Barges and
`Faulk (1981). The amnion was cultured in Petri
`dishes containing RPMI 1640 medium with 10%
`newborn calf serum buffered to pH 7.4 with
`HEPES. The culture medium was changed when—
`ever the pH rose above 7.6. No antibiotics were
`added. The amnion was thoroughly rinsed in sterile
`PBS immediately before applying it to the leg ulcer.
`
`Extra-embryonic membranes were collected from
`A 10 x 5 cm strip of amnion was placed between
`women undergoing elective Cacsarian section at or
`463
`
`2. Frozen
`
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`
`
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`464
`
`BRITISH JOURNAL OF PLASTIC SURGERY
`
`two sterile nylon sheets, put in a sterile universal
`container and then rapidly frozen to — 25°C. It was
`kept at this temperature for up to 6 weeks and was
`defrosted immediately before use by warming the
`container to room temperature for 10 minutes. A
`swab was taken from each piece of amnion after
`defrosting, for bacteriological examination.
`
`3. Fresh
`
`5 x 5 cm squares of amnion were immersed in Petri
`dishes containing sterile PBS and were stored at
`4°C for a maximum of 24 hours before use. A
`sample of the PBS in each dish was taken for
`bacteriological examination.
`
`4. Lyophr'lised
`Pieces of amnion of varying sizes were laid onto
`trays of unsterile nylon netting and freeze-dried in
`a vacuum at —~ 30°C. Each piece was then placed in
`a universal container and irradiated with gamma
`rays (60,000 Rad). The amnion was stored in a cool
`dry place for up to 3 months. Reconstitution was
`by immersion in Ringer's solution for one minute,
`after spreading the amnion aseptically on a piece
`of paraffin gauze. The amnion was then applied to
`the leg ulcer without removing it from the paraffin
`gauze.
`All the patients had chronic unilateral or bilateral
`venous leg ulcers which had failed to heal over 6
`months despite intensive medical treatment. Ar-
`terial insufiiciency was excluded by clinical exami—
`nation of the peripheral arteries and, if no pulse
`was palpable, by Doppler ultrasound. Patients with
`diabetes were not included. The patients were
`admitted to hospital for elevation of the legs and
`bed rest. The ulcers were dressed for 48 hours with
`saline soaks. Clinically infected ulcers were dressed
`twice daily with eusol and paraffin until clean and
`then with saline soaks for 24 hours to remove any
`traces of the eusol and paraffin solution.
`Before the trial was started 80 randomisation
`cards. 20 for each treatment group, were shufl‘ied.
`On admission 3 card was drawn to place the patient
`in one of the four groups. If there were two ulcers
`on one leg or bilateral ulceration, each ulcer was
`allocated separately to one of the four treatment
`groups.
`Pieces of prepared amnion were applied to cover
`the whole of the unhealed leg ulcer. The amnion
`was covered with paraffin gauze and an occlusive
`dressing. This was left undisturbed for 5 days and
`the patient was confined to bed. The dressing was
`removed under general anaesthetic in the operating
`
`theatre at the time of the split skin grafting. The
`skin grafts were first inspected on the 5th postop-
`erative day and the take of the graft was estimated
`by the following method: the borders of the graft
`and of the ulcer margins were traced onto a sterile
`transparent nylon sheet which was then laid onto a
`0.5 cm square grid. From this the area of the ulcer,
`which was equal to the area of the original graft,
`and the area of the graft which had taken were
`measured. The percentage take of the graft was
`then calculated.
`
`If there was no take of the graft, stored skin was
`applied 2 days later. In all other cases the graft was
`cleansed and redressed with paraffin gauze and an
`occlusive dressing. The patients remained on bed
`rest until the 10th day when mobilisation with
`below-knee supportive bandaging was started.
`The trial was approved by the local ethical
`committee and informed consent was obtained
`from each patient before admission to hospital.
`
`Results
`
`Twenty-seven patients with a total of 40 ulcers were
`treated in this trial. Their ages ranged from 47 years
`to 85 years (mean 65 years): 5 were male and 2!
`were female. The ulcers occurred on the left leg in
`11 patients and on the right leg in 8 patients: 8
`patients had bilateral ulceration. ln 5 patients there
`were two ulcers on the leg: each was treated as a
`separate ulcer.
`Eleven patients (1 7 ulcers) had clinically infected
`ulcers on admission which were dressed with eiisol
`and paraffin until clean followed by 24 hours of
`saline soaks before the application of amnion.
`Sixteen patients (23 ulcers) had clinically uninfected
`ulcers which were treated with amnion 48 hours
`after admission.
`Nine ulcers were treated with fresh amnion, 12
`with frozen amnion, 16 with lyophiliscd amnion
`and three with tissue-culture-maintained amnion.
`There is an imbalance between the groups as a
`result of the method used for randomisation. Forty
`ulcers were treated, thus using only half of the
`randomisation cards. Only continuation of the trial
`to 80 ulcers would definitely have given equal
`numbers in each group.
`Few patients received tissue—culture-maintained
`amnion because of problems which were found as
`the trial progressed. These led to the abandonment
`of this technique during the trial. It was difficult to
`maintain a steady pH of the tissue culture medium
`‘ despite the addition of bufl‘ers. Infection of the
`
`
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`

`'t‘
`
`THE HEALING OF CHRONIC VENOUS LEG ULCERS WITH PREPARED HUMAN AMNION
`
`465
`
`medium occurred on several occasions despite
`careful attention to sterility. Fifty-five pieces of
`amnion were maintained by tissue culture, of which
`four became infected with Staphylococcus albus
`within 2 days of starting incubation and two were
`infected by the 6th day; four grew Aspergillus sp. on
`the 5th day.
`One extra-embryonic membrane was collected
`from a mother whose baby developed a pustular
`rash around the neck, face and perineum within 15
`minutes of delivery. The rash cleared after 24 hours
`without treatment. Nose, throat and skin swabs
`from the baby yielded no growth but the amnion
`grew diphthcroids after 5 days of incubation.
`None of the samples of PBS taken from the Petri
`dishes in the fresh group nor any of the swabs taken
`from the thawed frozen amnion grew any orga-
`nisms.
`The take of the skin grafts in each of the four
`groups is shown in Table l. Lyophilised amnion
`gave the best take of the skin grafts (mean 74.7%).
`Fresh and frozen amnion each achieved similar
`results (mean 56.7% and 53.3% respectively). The
`mean percentage take of graft in the three patients
`who received tissueculture-maintained amnion
`before its use was abandoned was 58.3%.
`The number of complete failures of the skin
`grafts to take was lowest in the lyophilised group
`(6.3%); 11.1% of the fresh group and 25.0"/o of the
`frozen group failed completely and needed the
`application of stored skin. Failure of the grafts to
`take prolonged the healing time and consequently
`delayed the patient’s discharge from hospital
`
`The time taken for the Skin grafts to heal fully is
`shown in Table 2. The mean healing time was 27.9
`days for fresh amnion, 25.0 days for frozen, 22.3
`days for lyophilised and 30.7 days for the three
`patients in the tissue-culture-maintained amnion
`group. The mean healing time in the lyophilised
`group was lower than in the other groups because
`of the good graft take.
`Most patients were discharged from hospital 2
`days after the grafts were healed but in three cases
`discharge was delayed by 6, 10 and 22 days for
`social reasons. Two patients were transferred to
`long-term residential care and their discharge was
`considerably delayed while these arrangements
`were made.
`
`Overall. lyophilised amnion gave the best results
`both in the take of the skin grafts, and consequently
`low healing times, and in the lowest number of
`complete failures of the grafts to take. Although
`these differences are not statistically significant,
`lyophilised amnion was the easiest to use and store
`and was at least as effective as the other preparations
`of amnion in promoting wound healing.
`
`Discussion
`
`Leg ulcers have been estimated to affect about 1%
`of the population (Callam er a1., 1985). Treatment
`is generally unsatisfactory and many patients
`remain unhealed despite venous surgery and pro-
`longed medical treatment. Although it is generally
`considered to be a disease of the elderly, a recent
`survey has shown that over one-third of patients
`
`Table l The percentage take of the skin grafts on the 5th postoperative day
`
`
`Amnion preparation
`Mean (x)
`Fresh
`0 20 25 50 50 90 90 90 95
`56.7
`Frozen
`O 0 0 20 SD 50 775 75 90 90 95 95
`53.3
`Lyophilised
`0 20 4O 50 75 75 90 90 90 95 95 95 95 95 95 95
`74.7
`Tissue-culture-
`50 50 75
`58.3
`maintained
`
`
`
`Table 2 The time in days for the skin grafts to heal fully
`
`
`Amnian preparation
`Mean (days)
`Fresh
`13 l9 19 22 23 35 36 37 47
`27.9
`Frozen
`15 15 15 16 I7 24 28 30 32 35 36 37
`25.0
`Lyophilised
`10 13 13 15 16 17 17 20 24 24 25 27 28 32 37 39
`22.3
`Tissuetulture-
`27 28 37
`30.7
`maintained
`
`
`
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`

`

`n.__
`
`466
`
`have ulceration before the age of 50. (Callam et al.,
`1987).
`Amnion was first used on venous ulcers in 1913
`
`by Stern and has been used intermittently for this
`purpose since. It was found to have angiogenic
`properties when implanted in the thigh of patients
`with peripheral vascular disease (Troensegaard-
`Hansen, 1956) and when used on burns (Kirsch-
`baum and Hernandez, 1963). The development of
`profuse granulation tissue in leg ulcers after the
`application of amnion has been demonstrated both
`histopathologically
`and
`immunohistologically
`(Faulk er al., 1980). Angiogenic and growth-
`promoting factors in amnion have now been
`identified (Burgos, l983)artd partially characterised
`(Burgos. 1986). These factors appear to be high
`molecular weight complexes (molecular weight
`greater than 100,000) formed by active factor and
`carrier protein. The angiogenic factor has a molec-
`ular weight of between 400 and l 100 and is thought
`to be a peptide. Other factors with a molecular size
`between 500 and 30,000 molecular weight have also
`been identified. The factors are stable when frozen
`or lyophilised (Burgos, I983).
`The first clinical studies on the healing properties
`ofamnion used amnion maintained by tissue culture
`(Bennett et (11., 1980). In this trial we have used this
`technique on a larger scale and have found several
`drawbacks. Laboratory facilities are needed and
`close supervision .of the incubating process is
`necessary to watch for infection and excessive pH
`changes. The medium needs changing approxi-
`mately every 2 days to rectify pH changes and there
`is an increased risk of infection at this time. We do
`not know how long the amnion can be maintained
`in this way and still possess active angiogenic and
`growth-promoting factors. Amnion is known to
`survive well in culture for 2—3 weeks (Burgos and
`Faulk,
`I981) but
`the mitogenic properties of
`placental growth factors have been shown experi-
`mentally to fall to 35% of their original activity
`level after 8 days‘ incubation at 37°C (Burgos,
`1987).
`Although treatment with tissue-culture-main-
`tained amnion gave a fair take of the skin grafts
`(mean 58.3%)
`in three patients.
`its use was
`abandoned because of infection and the difficulties
`encountered in its preparation. We did not therefore
`investigate the possible decrease in activity of the
`angiogenic and growth-promoting factors.
`The patients were admitted with ulcers in varying
`degrees of cleanliness. We standardised their
`treatment into two groups, ensuring that the ulcers
`
`BRITISH JOURNAL OF PLASTIC SURGERY
`
`which were treated with eusol and paraffin had 24
`hours of saline soaks before the application of
`amnion so that any traces of hypochlori to or parafiin
`were eliminated. Although bed rest promotes
`healing of venous ulcers we feel that any advantage
`of the longer period of bed rest which the patients
`with infected ulcers were given was counterbal-
`anced by the poor state of the ulcers on admission.
`This trial was discontinued before the results
`reached statistical significance because concurrent
`research into the incorporation of the angiogenic
`and growth—promoting factors into synthetic dress-
`ings had been successful encugh to warrant clinical
`trials (Burgos. 1987). We are continuing to evaluate
`the use of a synthetic dressing impregnated with
`active factors (Burgos, in press).
`This trial shows that lyophilised amnion is the
`preparation of choice as it is efiective in promoting
`a high graft take and it is easy to store and to use.
`It has the disadvantage that although the prepara-
`tion is technically easy, a freezedrying machine is
`needed. Access to a gamma ray source for sterilisa-
`tion after lyophilisation is also necessary. lts storage
`time. however, appears to be indefinite as the
`factors in lyophilised amnion are known to be
`stable. It can thus be kept at room temperature and
`easily available in the ward. Reconstitution imme-
`diately before use is simple.
`
`Acknowledgements
`
`We are grateful to Mr J. E. Bowen and Mr N. M. Breach for
`permission to include their patients in this trial.
`D. J. Ward was supported by a grant from British Petroleum
`pic.
`H. Burgos was supported by grants from the East Grinstead
`Medical Research Trust and Johnson & Johnson Ltd.
`
`References
`
`Bennett, J. P.. Matthews, R. N. and Faulh, W. P. (1980).
`Treatment of chronic ulceration of the legs with human
`amnion. Lancet, I. ”53.
`Barges. H. ( 1983). Angiogenic and growth promoting factors in
`human amniochorion and placenta. European Journal of
`Clinical Investigation, )3. 289.
`Burgos. H. (1986) Angiogenic factor from human term placenta.
`Purification and partial characlerisation. European Journal of
`Clinical lrruesligaubrr, I6. 486.
`Burgos, H. (1987) Incorporation and release of placental growth
`factors in synthetic medical dressings. Clinical Materials. 2.
`133.
`Burgos. H. (in press). The purification and clinical application
`of angiogenic and growth factors from human placenta and
`endometrium. Possibleautocrine-paracrine role. In : Genscev,
`0.. Klopper. A. and Besconsfield, R. (Eds) Human Placenta
`as a Model nnda Source. New York: Plenum Publishing Corp.
`
`MTF Ex. 1016. MM
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`MTF Ex. 1016, pg. 4
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`

`
`
`THE HEALlNG OF CHRONIC VENOUS LEG ULCERS WITH PREPARED HUMAN AMNlON
`
`467
`
`Barges. H. and Fanlk. W. P. (1981). The maintenance of human
`amniotic membranes in culture. British Journal of Obstetrics
`and Gynaecology, 88, 294.
`Callam. M. J.. Ruekley, C. V. Harper. D. R. and Dale. J. J.
`(1985). Chronic ulceration of the leg: extent of the problem
`and provision of care. Brltllth Medical Journal. 290, 1855.
`Callarn. M. J., Harper, D. R., Dale, J. J. and Ruckley, C. V.
`(1987). Chronic ulcer of the leg: clinical history. British
`MedicalJournal. 294. 1389.
`Dino. B. R.. Eufeurio, G. G. and De Villa, M. S. (1966). Human
`amnion: the establishment clan amnion bank andits practical
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`Association. 42. 356.
`Fault. W. P, Matthews, R. N., Stevens. P. 1.. Bennett. J. P”
`unmet}. end Hal, 3. L. (1980). Human amnion asan adjunct
`in wound healing. lancer, 1, 1156.
`Kirschbaurn. S. M. and Hernandez, H. (1963). Use of amnion in
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`in Plastic Surgery. Amsterdam: Excerpta Media.
`Mottltews. R. N.. Faulk. W. P. and Bennett. J. P. ([982), In:
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`cut : Appleton-Century—Crofts.
`Notes, E.. Hirsebowltz, 3., Karev, A., Levi. J. and Mahler, D.
`(1975). The use of lyopbilised amnion to treat burns and skin
`defects. Hare/hall. 88. 265.
`Snbelln, N. (1913). Use of ietal membranes in skin grafting.
`Medical Record: anew York. 8. 478.
`Sterling, J. A. (1956). Use of amniotic membranes to cover
`
`surface defects due to flame burns. American Journal 0]
`Surgery. 91, 940.
`Stern. M. (1913). The grafting of preserved amniotic membrane
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`Journal ofthe American Medical Association, 60. 973.
`humped-Hansen, E.
`(1956). Amnion implantation in
`peripheral vascular disease. British Medical Journal. 2, 262.
`Unger, M. G. and Roberts, M. (1976). Lyophilised amniotic
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`
`The Authors
`
`D. J. Ward, FMS. Clinical Research Fellow and Honorary
`Senior Registrar. Blond Mclndoe Centre for Medical Re-
`search, Queen Victoria Hospital. East Grinstead. West Sussex
`RHI9SDZ.
`J. P. Bennett, FRCS. Consultant Plastic Surgeon. Queen Victoria
`Hospital.
`H. Brrrgos. PhD. Research Fellow. Blond Mclndoe Centre for
`Medical Research.
`J. Fabre. MB, BS, B.Med.Se.(Hons). PhD, Professor and
`Director of Research, Blond Mclndoe Centre for Medical
`Research.
`
`Requests for reprints to Mr Ward.
`
`Paper recein 10 May l988.
`Accepted 19 December 1988 after revision.
`
`MTF Ex. 1016. pg. 5
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`MTF Ex. 1016, pg. 5
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`