`DRES ING MARTIN
`
`AMNI
`
`AS A TEMPORARY WOUND
`
`, M.D., THOMAS j. KRIZEK, M.D., FALLS, NEAL KOSS, M.D., and
`
`JONATHON L. SAMBURG, D.D.S., New Haven, ConneCtiCUC
`
`TEMPORARY BIOLOGIC CLOSURE of a burn or an
`open wound has often proved to be a lifesaving
`measure. Various substances, such as allograft and
`xenograft skin, have been used in an attempt to
`obtain such closure.
`In so doing,
`they decrease
`pain, decrease protein loss,
`reduce evaporative
`heat and water loss from the wound surface, and
`prevent further contamination. Another function
`desirable in a biologic dressing is its ability to
`decrease existing bacterial grewth. Allograft skin
`performs this function better than does xenograft
`skin.
`
`amniotic membranes have been
`Recently,
`evaluated experimentally as temporary biologic
`dressings. In a series of 50 rat bums infected with
`10" Pseudomonas aeruginosa organisms, amniotic
`membranes were shown to be one thousandfold
`more reflective than split
`thickness human skin
`grafts in decreasing the bacterial
`level
`(10). By
`using a diflercnt experimental model, amniotic
`membranes were compared to split thickness iso—
`graft skin, split thickness allograft skin, and split
`thickness xenograft skin as
`to their
`respective
`abilities at decreasing bacterial levels in full thick—
`ness defects created on the backs of rats (12). The
`amniotic membranes were found to be equal to
`isografts and superior to both allograft and xeno-
`graft skin. Based on these two reports, clinical
`trial was indicated to evaluate amniotic membranes
`as a temporary biologic dressing for Open wounds.
`MATERIALS AND METHODS
`
`Preparation of amniotic membranes. Fresh amniotic
`membranes were obtained at the time of delivery
`from seroncgalivc mothers, who had no history of
`either premature rupture of the membranes or
`endometritis. Heavily meconium stained or foul~
`smelling membranes were discarded. The mem~
`branes were aseptically removed from the placenta,
`with no attempt being made to separate them into
`amnion and chorion.
`'With the use of an aseptic
`technique,
`the membranes were passed through
`From the De artmcnt of Surgery (Plastic), Yale University Selmol
`of Medicine,
`cw Haven.
`
`
`
`
`
`
`
`
`In all full thickness defects, the amniotic mam
`branes were placed on the wound with the ulterior:
`against
`the granulating bed and the smooth,
`glistening amnion surface away from the wound.
`The membranes were left either exposed or cov-
`ered with a protective dressing to prevent dis
`lodgment. The membranes were changed routinel?
`every 48 hours. Prior to application of the first
`amniotic membranes and at
`the time of each
`dressing Change, specimens were obtained from
`the granulating bed for qualitative and quantitative
`bacterial analysis. The method of bacterial analysis
`has been described previously (9).
`Partial thickness wounds were handled in two
`different ways. Early in the study, the membranes
`were placed on the partial
`thickness wounds:
`chorion against wound, and the membranes were
`changed every 48 to 7’2 hours.
`In the latter
`part of the study, the amnion was placed against
`the surface of the wound, and the membfafle
`was allowed to remain in place until it: separated
`spontaneously.
`904
`
`four rinses of sterile saline solution, one rinse of
`0.025 per cent sodium hypochlorite solution, and
`an additional four rinses of saline solution. In each
`rinse,
`the membranes were agitated thoroughly to
`dislodge any "clot or foreign material which might
`be present. After preparation,
`the membranes
`were refrigerated in individual sterile containers at
`4 degrees C. The membranes were cultured
`routinely at weekly intervals. The Cultures were
`uniformly sterile. No material was used after six
`weeks of storage.
`Clinicat’ we of amniotic membranes. Fifty open
`wounds in need of temporary biologic dressings
`were Chosen for study. These included both full
`thickness and partial thickness defects of themml
`and nonthermal origin. The application of biologic
`dressings was begun in full thickness dtft’cts only
`after all esoliar had been removed. If antibacterial
`agents had been used topically prior to the insti-
`tution of biologic dressings,
`the medication was
`removed thoroughly by immersing the patient in a
`Hubbard tank.
`
`MTF Ex. 1020, pg. 1
`
`
`
`
`
`JONATHON L. SAMBURC, D.D.S., New Haven, Connecticut
`
`four rinses of sterile saline solution, one rinse of
`0.025 per cent sodium hypochlorite solution, and
`an additional four rinses of saline solution. In each
`rinse,
`the membranes were agitated thoroughly to
`dislodge any clot or foreign material which might
`be present. After preparation,
`the membranes
`were refrigerated in individual sterile containers at
`4 degrees C. The membranes were cultured
`routinely at weekly intervals. The cultures were
`uniformly sterile. No material was used after six
`weeks of storage.
`‘
`Clinical use If amm'alz‘c membranes Fifty open
`wounds in need of temporary biologic dressings
`were chosen for study. These included both full
`thickness and partial thickness defects of thermal
`and nonthermal origin. The application of biologic
`dressings was begun in full thickness defecu onlyr
`after all esohar had been removed. If antibacterial
`agents had been used topically prior to the insii
`tution of biologic dressings,
`the medication was
`removed thoroughly by immersing the patient in a
`Hubbard tank.
`
`TEMPORARY BIOLOGIC CLOSURE of a burn or an
`open wound has often proved to be a lifesaving
`measure. Various substances, such as allograft and
`xenograft skin, have been used in an attempt to
`obtain such closure. In so doing,
`they decrease
`pain, decrease protein loss,
`reduce evaporative
`heat and water loss from the wound surface, and
`prevent further contamination. Another function
`desirable in a biologic dressing is its ability to
`decrease existing bacterial growth. Allograft skin
`performs this function better than does xenograft
`Skin.
`
`been
`amniotic membranes have
`Recently,
`evaluated experimentally as temporary biologic
`dressings. In a series of 50 rat burns infected with
`108 Pseudomonas aeruginosa organisms, amniotic
`membranes were shown to be one thousandfold
`more ellcctive than split
`thickness human skin
`grafts in. decreasing the bacterial
`level
`(10). By
`using a different experimental model, amniotic
`membranes were compared to split thickness iso»
`graft skin, split thickness allograft skin, and split
`thickness xenograft skin as
`to their
`respective
`abilities at decreasing bacterial levels in full thick~
`ness defects created on The backs of rats (12). The
`amniotic membranes were found to be equal to
`isogral‘ts and superior to both allograft and xeno—
`graft skin. Based on these two reports, clinical
`trial was indicated to evaluate amniotic membranes
`as a temporary biologic dressing for open wounds.
`MATERIALS AND METHODS
`
`In all full thickness defects, the amniotic mem-
`branes were placed on the wound with xhe chorion
`against
`the granulating bed and the smooth,
`glistening amnion surface away from the wound»
`The membranes were left either exposed or cox-u
`cred with a protective dressing to prevent dis~
`lodgment. The membranes were changed routinely
`every 48 hours, Prior to application of the first
`amniotic membranes and at
`the time of each
`dressing change, specimens were obtained from
`the granulating bed for qualitative and quantitative
`bacterial analysis. The method of bacterial analysis
`has been described previously (9).
`Partial thickness wounds were handled in two
`different ways. Early in The study, the mei'nhrmzcs
`were PlaCCd on the partial
`thickness wound;
`chorion against wound, and the membranes were
`Changed every 48 to 72 hours. In the latter
`part of the study, the amnion was placed against
`the surface of the wound, and the membrane
`was allowed to remain in place, until it separated
`spontaneously.
`904
`
`Preparaiion (j amniotic membranex. Fresh amniotic
`membranes were obtained at the time of delivery
`from seronegative mothers, who had no history of
`either premature rupture of the membranes or
`endometritis. Heavily Ulcconlum stained or foul—
`smelling membranes were discarded. The mem-
`branes were aseptically removed from the placenta,
`with no attempt being made to separate them into
`amnion and chorion. With the use of an aseptic
`technique,
`the membranes were passed through
`From the De artmem of Surgery (Plastic), Yale University School
`of Medicine,
`cw Haven.
`
`MTF Ex. 1020. pg. 2
`
`
`
`
`
`
`
`
`
`
`
`Robson at (1L: AMNIO’I‘XC MEMBRANES AS TEMPORARY WOUND DRESSING 905
`c membranes were evaluated in the
`related this failure to control bacteria to the poorer
`wounds bf‘adherence to the wound,
`¥
`‘
`adherence to the wound with xenografts.
`1,3 Eh}; mesfin Patient discomfort, and by control
`Amniotic membranes have few of the disad-
`, (21516 :«owth‘ In the pardal thickness defects,
`‘
`vantages of cadaver allografts and xenografts.
`”1 hui‘cjlullcils wefé 1301 measured.
`They are readily available and are of no cost to
`mm" ‘
`'
`thc'patient. As shown both in the previous ex-
`perimental studies and in the clinical use,
`they
`adhere well and obtain a biologically closed
`wound. Histologically,
`they are quite similar to
`skin. The membranes are made up of two layers,
`the uninion and the chorion. The inner layer of the
`amnion is smooth and glistening, being cemposed
`of cuboidal cells. Its outer surface is mesenchymal
`connective tissue. The chorion has mesenchymal
`connective tissue in contact with the amnion
`and an external
`layer of transitional epithelial
`cells. Pigeon (7) has stated that, since the amniotic
`membrane is formed from the ectoderm of the fetus,
`it is like an extension of the skin of the baby.
`Preparation of
`the amniotic membranes in
`dilute sodium hypochlorite solution was chosen
`after a careful review of the literature. Dino and
`his associates
`(4) have reported the testing of
`many agents
`to sterilize the membranes. We
`believed that any substance chosen should not, in
`itself, be antibacterial or so chemically powerful
`as
`to change the biologic effectiveness of the
`amniotic membrane.
`
`ounds studiCCi were divided among 42
`“SH/Ts
`““'.50W. defects and eight. partial
`thickness
`{all ”3161,:ch '
`tiC membranes adhered to all
`moms.
`. mmO q
`f
`l
`~
`1.
`h ‘5“
`.
`.
`,, rflawless, o i leir ( opt
`. e lrmlarly, in all
`minim :in was markedly relieved as soon as the
`wannfiixfig (3;ch placed onto the wounds,
`wit: :2ng of the full
`thiclfneéis wounds did the
`lu‘u‘lcrizll
`(301111t Increase While Gang treatsd WJth
`mmimic membranes.
`in several of the wounds,
`limngmic membranes were (toinparcd simultane-
`;;ugl\’ with other biologic dressmgs, and in these
`gaxiziems,
`the decrczise‘
`in bacterial count wag
`«qua! to allogral‘t Slim and superior to xenograft
`skin.
`Amniotic membranes provided excellent dress—
`gags for partial thicknesr defects. They provided a
`“rammivc covering, beneath which epithelializa-
`lion could occur. The mos: striking features in the
`ummires of partial thirkneas were. the immediate
`.ulhcrencc and relief of pain. The amniotic mem-
`branes proved equal
`to standard dressings when
`mrd on partial
`thickness burns, donor sites, and
`«m the face after dermnbmsion.
`lilSilL’SfilON
`
`The COnCCPC of temporary biologic dressings for
`”5“ 9n burns, popularized by Brown and his
`"Wmmcs l2: 3}, has been extended to use on all
`Warn wounds. Allograft $211 and xenograft skin,
`5:33:31€11???“ng most criteriei
`for satisfactory
`“mama {0:531:1135: hfwc their limitations. Cadavers
`,3“. general ah Digital! use arc in limited supply in
`quirt‘d fig;
`05131131, Because personnel ore re-
`m“, six ph ilficurcmem, Baxter (I? has estimated
`gm patienggcian how‘s and a hospital cost of $225
`Xi‘riograh “Seedcd'io use cndavcr allografts.‘
`mm l351V2iilqgiin3
`Initially dillicult
`to obtain,
`“Wm; {or if:
`L In fresh,
`lyphohzed, and frozen
`Xz’zmgrzzftsc out $20 per square foot, Hewever,
`‘imwh, Ra 0 “0’; Uniformly control baccerlal
`““lkfl‘ml thf’aport and his (to-workers (8) have
`”W! 241mm, xenogl‘afts left
`in place for longer
`underlying; b.0361} x rm not control the growth of
`“mills
`‘
`.
`In the patients in whom
`z“"Wmsl
`71
`dressings were compared simul—
`'
`ll‘crim.
`t1: piescnt'study, xenografts proved
`'
`st “a logralts at decreasmg bacteria
`em and his Colleagues (14) have
`
`‘.
`
`A complete review of the preferences of various
`authors as to the use of chorion alone, amnion
`alone, or
`the combination of intact amniotic
`membrane has been presented previously (10).
`We prefer to use the entire amniotic membrane
`because it eliminates the need to divide the mom.
`branes at the time of preparation. Also, the thicker
`intact membrane appears not to dessicate on the
`wound as quickly as does amnion alone. Most
`importantly,
`it has been shown that bacterial
`growth is decreased most effectively when the
`temporary biologic dressing achieves an initial
`take onto a granulating wound (11). The lack of
`effectiveness of xenograft has been demonstrated to
`be related to its inability to cause this initial take.
`Although, the only studies on amniotic membranes
`showing this initial neovascularization were done
`in rats by Douglas and his colleagues (5), results of
`their experiments conclusively demonstrated vas-
`cular penetration. Their studies were perforined
`using chorions. Therefore,
`in the present series,
`we have elected to place the choriomc curfew
`against the wound to allow the revasculanzation
`to proceed into the less dense tissue. For the smile
`reasons, we have elected in the last few partial
`thickness injuries to reverse the membrane and
`place the amnion against the wound. In partial
`
`
`
`MTF Ex. 1020. pg. 3
`
`
`
`
`
`Valuing 136
`
`906 Sargon, Gynecology e’yObstetrics - June1973 -
`thickness defects, vascularization is not desirable,
`and it
`is hoped that
`the mere dense uniform
`cuboidal surface of the amnion would discourage
`penetration. Indeed, Graham (6), using amnion
`alone on partial
`thickness defects, has shown
`that an initial take dees not occur.
`SUMMARY
`
`After demonstration of the value of amniotic
`membranes as temporary biologic dreesings in
`experimental models,
`50 patients with open
`wounds were treated with amniotic membranes.
`In full
`thickness injuries,
`the membranes gained
`immediate adherence, decreased pain, and de—
`creased bacterial contamination in the wounds.
`In partial
`thickness wounds,
`they afforded ex-
`cellent cover, while allowing re-epithelialization to
`occur. Based on these results, a more widespread
`clinical trial is indicated in burns and other open
`wounds.
`
`REFERENCES
`1. BAxnzn, C. R. Homografts and heterografts as a
`biologic dress‘
`in the treatment of thermal injury.
`Presented at
`he First Annual Congress of The
`Society of German Plastic Surgeons
`in Munich,
`Germany, Sept. 28, 1970.
`2. BROWN,J. B., an, M. P., RANDALL, R, and Lu, M.
`Postmortem homografts as “biological dressings” for
`
`10.
`
`11.
`
`12.
`
`13.
`
`
`
`
`
`
`
`3)
`
`mtensive burns and dcnudcti areas. Ann. 3mg“ 193
`138: (318.
`.
`.
`BROWN, 1. B., and Ntd'kgwxu, in Massive “Pairs
`bums with thick spin—5km grafts. Ann. Surg., 194?l
`115: 658.
`Dem, B. It, EUPEMIO, G. 0., and DEVILLA, M. S
`Human amnion;
`the establishment of an amniu'
`bank and its practical applicatinns in 5mg”? ‘1
`Phillip. Med. Assam, 1966, 42; 357.
`’1 J-
`DOUGLAS, B“ C(WWAY, U. STARK, R. 3., and 09103
`The fate of homologous and heterologoua ChDfiDDi.
`transplants as observed by the transparent mm“
`clmmber technique in the mouse. Mast. Remus;
`Surg., 1954,13:125.
`'
`GRAHAM. W . C. Personal communication.
`PiGEGN, j. Treatment of second degree burrs with
`amniotic membranes. Can. Mcd. Axum. j., 1960
`83: 844.
`‘
`RAPPAPGRT, 1., PEPINO. A. T., and Dmmex‘ w
`Early use of xcnngrafts as a biologic dressing in bun;
`trauma. Ami
`. Surg., 1976, 120; M4.
`Ronsorc, M.
`., and Kansans}. P. Bacterial quanufi.
`cation of open wounds. Milit. Meet, 1969, I34: 19,
`Reason, M. (2., and Knizen, '12}. The effect of human
`amniotic membranes on the bacterial population of
`infected rat bums. Ann. Surg., in press.
`idem. Predicting skin graft survival. }. Trauma, in
`ress.
`“1., Smmtmo, j. L., and Kazzxx, T_ J,
`pRUBSQN, M.
`Quantitative comparison of biologic drawings. Surg.
`Forum, 19?2, 23: 503.
`Suvnnsmm, R, Cunnem. P. V92, and Mumm, A. M.
`Evaluation of fresh, viable porcine cutaneous xeno-
`graft as a temporary bum wound cover. Annual
`Progress Report, U. 5. Army Institute of Surgical
`Research, 19?0~71.
`
`MTF Ex. 1020, pg. 4
`
`
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