Use of Dry Human and Bovine Amnion as a Biological Dressing (2)

7/26/2019 Use of Dry Human and Bovine Amnion as a Biological Dressing (2)

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Use ofDry Human and Bovine Amnionas a Biological DressingT. Viswanatha Rao, MS, V. Chandrasekharam, MS

\s=b\A new method of drying and sterilizing the amnion extendsthe life of the productto more thannine monthsand enables ittobestored at room temperature.The bovine amnion isuniqueandthe first of its kind, there being no comparable series inthe literature, to our knowledge.Bovineamnionhas a large surfacearea and canprovide bulksupplies. The membrane as preparedhas beentested in 12 rabbitsexperimentallyand in 70 patientsofdifferent ages with a variety of surgicallesions,mainly burns.Morethan150 applications of thedriedamnion havebeen made as thesole dressingfor thesepatients.The results indicatethatamnion,both human andbovine, is quitesafe and effectivea s a biological

dressing.The dried material retains all thephysical and biologicalcharacteristicsof the freshmembrane.(Arch Surg1981; 116:891-896)

The idea of fetal membranes as a substitute for skinwas firstsuggested by Davis.1 Thefirstclinicaltrial iscredited to Sabella.2 Soon it was realized that these membranes were useful as temporarybiologicaldressings only.Johnson3suggested the use of bovine allantois as a sourceofbiologicaldressing. Chao et al4 were the first to use thedried andalcohol-preserved amniotic membrane for crani-otomy wounds to cover extensive durai defects and to

prevent adhesions. They observed severe reactions whenchorion was retained for a short time before application.Troensgaard-Hansen5 used amniotic grafts for the treatmentof chronicnonhealing ulcers.Allografts of skin fromdonors and cadavers were popularized by Brown et al.6Silvettiet al7suggestedthe use of xenografts from bovineembryo skin.

Interest in fetal membranes has been revived recently,

Accepted for publication Oct7, 1980.From the Department of Pediatric Surgery, Andhra Medical College,

Visakhapatnam, India.Reprint requests to Department of Pediatric Surgery, Andhra Medical

College,Visakhapatnam 530002, India (Dr Rao).

with multiple reports having been published in variousjournals. Human amniotic membrane has been used in allthese studies. The main drawback of these membranes

appears to be the difficulty of assuringadequate suppliesandthedifficulty of theirpreservation. Dino et a l8 studiedthe effect of human amniotic membranes in burns. Theydescribed the method of processing the membrane andsuggested theorganization of an amnion bank.

The earlier attempts at preservation of amnion describedby Chao et al seem to have beenabandoned, as no

further reports are

available of their methodshavingbeenpursued.Dinoet al havepreservedtheamnion at 4 Cafterrepeated rinses in saline solution. When kept in salinesolution at room temperature, 100% contamination of the

specimens occurred within 48 hours. Addition of 0.25%sodium hypochlorite to the saline solution keptspecimensfree of infection for one month. Addition of200,000 unitsof crystalline penicillin improved the viability up to sixweeks, especially if stored at 4 C. This method is verypopular and is extensively used by most workers in thefield.

Wehave attempted asimplertechnique that eliminatestheneedfor acostly amnionbankandthatenablesstorageat room temperature. The twin problems of preservationby a simple technique andprovision of adequate supplieswhen most needed will be solved by (1) preserving theamnionin thedry stateand (2)using thebovineamnion toprovide bulk supplies. This communication recounts ourexperiences with these two products.

Table 1 gives the characteristics of the two types ofamnion. Thelarge surface of the bovine amnion is worthnoting. Our figure for the human amnion is at variancewith that ofTroensgaard-Hansen,5 whoclaimsonly 52 sqcm (8 sq in) of amnion as being available from a singleplacenta.

The normal requirement of biological dressing for 50%

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Fig 1.Bovineamniotic sac reconstitutedby fillingwithtap water.Notelargesurface of sac. It hadto befilled with 15 Lof tap waterto distend it. It has been folded for convenience.

Fig 2.Dried amnion sealed in polythene covers.

burns in an adult has been estimated to be about 6,000 sqcm.9 Such large quantities of human amnion will bedifficult to procure, especially if the amnion has to bechangedevery 48hours. Allograftsandxenografts of skinare not available in many parts of the world, whereasamnion, both human andbovine, is availableuniversally atalmost no cost.

SUBJECTS AND METHODS

Freshplacenta isprocured at normal, healthy deliveries fromwomenwithout ahistory ofprematureruptureof membranes.Theamnion isseparated from thechorion and the fetal surfaceof theplacentaup to a shortdistance from the implantation of the cord,care being taken not to include Wharton's jelly as it preventsproper drying. The membrane is washed insaline to remove clotsanddebris.Finally,it is rinsedin salinesolutioncontaining alittlebleachingpowder. The membrane becomes bleached as well assterilizedby this method. It is now spread on a plastic sheet andallowed to dry in the open air. It dries within a short time andadheres to the plastic sheet. The membrane can easily be separated from theplastic sheet. It is then sealed inpolythene coversand labeled. The method of processing and drying the bovineamnion is identical (Fig 1).

The dried and sealed covers (Fig 2) containing amnion aresterilizedby(1) ultraviolet(UV)rays or(2) y rays. Forroutineuse,UV sterilization was quite adequate. One hundred packets ofamnion weresterilizedby y radiation. Randomsamplesof amnionwere testedbacteriologically forsterility.

ExperimentalAnimals

Twelve rabbits weighing 900 to 1,400 g were given a uniformdeep burn of 7.6 x 2.5 cm (3 X 1 in)under ether anesthesia. Fivedays later, escharectomy was followed by the application ofamnion(bovine only)to half of thewound,the other half being leftopen as acontrol. The amnion was not suturedto thewoundedges;it was simply allowed to adhere to the wound surface. Repeated

inspectionsof the wound were made at intervalsof fivedays each

until the healing was complete. Cultures were taken from thewound surfacesduringtheseinspections. Noother treatment wasgiven to the animals.

Clinical Subjects

Seventypatients with varioussurgical lesions, 58of them fromburns, are thesubjects of thisstudy. These 70patients had morethan 150 applications of the amnion. Thedetailed particulars ofthepatients aregiven in Tables 2through 4.

Superficial Burns.The wound was prepared by excision of blebsand slough. It was washed with sterile saline solution. Amnion,human or bovine, was applied to the raw surface and allowed todry. Except in the first tenpatients, only the dried and sterilizedamnion was applied to wounds.Nosutures or retainingdressingswereapplied.Localantibiotic creams were avoided.Antibioticsfora systemic effect and intravenous fluids were administered asneeded.

Deep Burns.The routine just described was modified slightly.Immediately afteradmission, as muchof the area aspossible wascovered with amnion without making any effort to assess thedepth of the wound. Blebs and overhanging skin andobviouslydead tissue wereexcised. These areas also werelatercovered withamnion. Escharectomy was performed at the earliest date afterrenalfunction wasreestablished,

usuallyaroundthe fifthday. The

raw area left after escharectomy was covered with amnion. Inextensiveburns,escharectomy wasperformedin stages.Exceptincases with gross local sepsis, antiseptic lotions were not usedroutinely. The amnion was changedonly when signs of infectionwere present or when the membrane showed signs of autolysis.

Pressure Sores.Because of gross infection in these cases, thewound was thoroughly cleansed with antiseptic lotions usingpovidone-iodine(Betadine), and slough wasexcisedprior to applicationsof the amnion. In deepexcavatingulcers, amnion was alsopacked into the depths of the ulcer. The dressing was changedrepeatedlyat least once daily.

RupturedMeningoceles.The siteof CSF leak was identified andthe area wascoveredwithamnionafterthe wound w as cleaned. In

case of persistence of the leak or in postoperative cases with



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Table 1.Characteristics of Amnion

Human Bovine

Surface area,"sq cm 1,600 6,000-7,500

Shape Spherical CylindricalThickness, /im 10 15

Histologie appearance

Single layer ofcuboldal

epithelium

Single layer ofcuboidal

epithelium

'Surface area is calculated as circumference x length or height.

Table2.Types of Cases

Amnion

Human

Fresh DryBovine,

DryOnly TotalHumans

Burns 15 32 58

Skin graft donor sites

Ruptured meningocelesPressure sores

Total 11 20 39 70Rabbits 12 12

Table 3.PatientCharacteristics: Age and Sex

Sex

Age Total

0-1 yr 11 16

1-4 yr 19

5-8 yr9-12 yr 15

Adult 11

Total 47 23 70

The male-female ratio is 2:1.

Table 4.Surface Area and Degree of Burns

Area, % Superficial Deep Mixed Total

10 16 23

20 10

30 10

40

50

>50

Total 29 20 50s

Data for the remaining eight cases are missing.

wound sepsis only, the amnion was changed after 24 to 48hours.

RESULTS

Experimental Animals

Thesafety and effectiveness of the bovine amnion hasbeenestablished. A ll of the rabbitssurvivedtheprocedure.The wounds healed within three weeks. The membrane

adhered to the wound surface and remained adherent

throughout theperiod ofhealing, even though it was notsutured and adressing was not applied. A single application was

enough.

Fig 3Burns induced in rabbit are covered with bovine amnion.Membrane isstill adherent on fifth day. Membrane-covered area,which iscleanand transparent, can be recognizedeasily. Controlarea is opaque and covered by eschar. Notethat no sutures holdamnion.

Fig 4.Baby with superficial burns dressed with amnion (human).

The objectives of ourstudy ontherabbits werelimitedtothe evaluation of thesafety,durability, and bacteriostaticactivityof the membraneand the effectiveness andadherenceof thebovineamnion. In all theserespects,the bovineamnionhasprovedsatisfactory. Cultures from theamnioncovering half of the wound were sterile while the control

area showed coagulase-positive Staphylococcus or Pseudo-monas pyocyanea organisms. The amnion-covered arearemainedtransparentandclean,whilethecontrol area was

opaque,dirty, andcoveredby dense eschar(Fig3).

Clinical SubjectsA singleapplication was adequate in superficial burns,

except in sodden areas like theperineum. The membraneadhered to the wound and remained adherent until the

regeneration was complete. In the case ofdeepburns,theamnion adhered both to theeschar and to the raw surface

following escharectomy. However, in view of the full-thicknesslossof skin and dueto thefrequent occurrenceof



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Fig 5.Child with extensive superficialburns treated with bovine amnion. On thefifthday, amnion is dried and adheres wellto wounds. In areas it peels off as healingtakes place from periphery.

Fig6.Boy with burns due to hot oil. Twomonths after accident, wounds weretreated with human amnion. Except forsmall area in front, rest of wound hashealed without need for grafting.

infectionin these cases,the amnionhad tobechanged oncein 48hours. In one case, the amnion had to bechanged tentimes in about eight weeks (Fig 4 through 6). All thesuperficial burns have healed without incident. None ofthem required skin grafting. We note that the arearequiringskingraftinghas also been reducedin the caseofdeep burns. Ashealing occurred from the periphery, onlypatchy islands in the center required grafting. Contractures and deformities are not eliminated by the use ofamnion, hence some form of splints is needed to preventthem.

There were no differencesbetween the various types ofamnionused. This indicatesthat the

processof

dryinghas

not affected the physical or biological properties of theamnion. Bovineamnion, whenapplied to either humans ortheexperimental animals, behaved like an allograft, andthis behavior was similar in the human amnion. Thisindicates that the bovine amnion is a safe and useful

biologicaldressing that is similar andperhaps superior toheterografts of skin. Apart from its large size, bovineamnion was more smooth andglistening, but itcrumpledmore easily andgotdisplaced from the wound more often,indicating less efficient adherence to the wound.

There were no ill effects from the use of irradiatedamnion. Irradiation did not appear to have affected the

physical or biological properties of theamnion, 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 reactions (local or systemic), all three types of amnion havegivenequallygood results.

2. With respect to prevention of fluid losses, amnionacted as an excellent vapor barrier by preventing furtherlosses of fluid from the wound bed because it adhered tothe wound surfacefirmly.

3. Patient morale was good in all cases.4. The intimate andclose bond between the wound and

the amnioneffectivelyprevented infection from the exte-

rior. In full-thicknessburns,infection was alreadypresentwithin the wound. In these cases,repeatedapplications ofthe membrane after early escharectomy controlled theinfection to a great extent.

The results in full-thickness burns were not uniformlyencouraging. However, surgical debridement and amnioncover over the area reduced the pain and shock. Thepatientsbecame more comfortableafter the application ofamnion, thus improving patient morale. Infection couldalso be reducedby the method.

Theperiod of convalescence was not significantly modifiedbythe use of amnion. Butsincethepatients were more

comfortableand

mobile, some

of them could be treated as

outpatients.Amnion was equally useful in nonthermal injury. In

ruptured meningoceles, amnion sealed off the wound andprevented the inevitable CSF leak. In pressure sores,infection was controlled in a short time and small ulcershealed by themselves. Larger ulcers were covered byhealthy granulations flush with the skin so that skingrafting could be undertaken.

Afterthewoundshealedin superficialburns,theamnionpeeled off from the surface of the wound. However, caseswith full-thickness losses showed healthy pink granulations that remained flush with the skin and to which the

amnionremained adherent. Thegranulations formed werenever exuberant.Changes ofdressing did not cause eitherdiscomfort or bleeding, which are common with otherforms of dressing.

COMMENT

Terminologyand Types of 'Amnion'

"Amnion" and "amniotic membrane" are terms used

loosely in literature. Most authorities in this field haveused"amnion"mainly. Colocho et al10 removed thechorionbeforeprocessingthemembraneand still called it "amniotic 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 thechorion,dried theremain

ing membrane, and called it "amnioplastin," which theypreserved in alcohol. Troensgaard-Hansen5 also removedthe chorion and called the final product "amniotic graft."Jenner,12 and later Dino et al," studied the effects of thetwo membranesseparately and concluded that there wasno difference between the various layers of the amnioticmembrane. Douglas et al1' noted vascular penetrationwhen chorion was used. Trelford et al14 did not find anyneovascularization when amnion alone was used. Theyhaveactuallysuggestedthatchorion may be antigenicandmay be rejected immunologically. Walker et al11 did notfind any immunological reactions from the amnion prepared by them. The role of chorion, according to them, isstill undefined. Troensgaard-Hansen5 has used thesmoothsurface of the amnioticgraft to face the woundsurface, ashethoughtthattheactiveprinciplesofthemembrane werepresentonly in the amnion.

The amnion preparedby us is similar to that preparedby most of the authors just mentioned, except that it isin the dried state. There have been no local reactions,

immunological or otherwise. There were no systemicreactions either. The membrane retained all thephysicalproperties of the fresh amnion, along with its beneficial effects on thepatient. We have observed, moreover,that in places where the chorion was retained, the membrane did not dry properly and the dried productremained sticky and waxy. It was also denser andopaque.

We find in the literature that Robson and Krizek15 didnot make anydistinctionbetween the variouslayersof thefetal membranes. They have used the complete amnioticmembrane.

Amnion seems

to satisfy all the criteria of an

idealbiological dressing: an effective vapor barrier, adherence,durability, bacteriostatic activity, protection from pain,andelasticity.16

Vapor Barrier

Amnion is an excellentvaporbarrier. Itresembles inthis

respectthestratum corneumepidermidisof theskin,whichis thenatural vapor barrier,17yet it hasonly one third thethickness of the stratum corneum epidermidis(10 to 15 umvs 30 to 60um).

It is important to note that amnion does not influencetheshiftof fluid in the extracellularspacefrom the plasmainto the interstitialcompartment. However,bypreventingevaporation from the wound surface, insensible losses,which may exceed 2 to 3 L in burns greater than 18% to40%,17 are prevented, thus reducing the quantity of fluidrequired. Furthermore, by preventing evaporation fromthewoundsurface,the temperatureregulation mechanismis not overstrained and the caloric requirement needed tomaintain the temperature is alsocorrespondingly reduced.Forthis to bereally effective, amnion should beapplied atthe earliest opportunity from the time of burning. Forprecise evaluation of the role of amnion in reducing thehypermetabolism and fluidandelectrolytebalance,further

studies are being made.

Physiological and Biological Properties

Amnion is a physiological-biological dressing since it isderived from the epiblast and is continuous with thesurface ectoderm of the fetus. It is, as Pigeon18 hasremarked, an "extension of thebaby's skin" and hence isthemost physiologicalof thebiological membranes. Silvet-ti et al7 have advocated the use of bovine embryo skingrafts. Structurally,amnion is made upof cuboidalepithe

lium on a

framework of mesenchyme. The epithelium,which is derived from the epiblast and which appearsbefore the embryonic plate is formed, remains in itsprimitive state and fails to differentiate further. Thisprimitive state, or less complete differentiation of theamnioticmembranes,is consideredto be the cause fortheirantibacterial activity.1' Immunological reactions are alsoconsidered to be absent because of this.

Thebacteriostaticactivity oftheamnion is said to be dueto the presence of antibodies,possibly allantoin and lyso-zyme.15 Itis not affectedby the process of drying or by theremoval ofthechorion, as experience shows. Thisindicatesthat the dried amnion retained all the antibodies in adormant state and that they were not destroyed by theprocess of drying, irradiation, or storage. Theactiveprinciplespresentin the amnionappearedto be stableunder alltheseconditions.The drymembraneadhered aswell as thefresh one andremainedadherentuntilwoundregenerationwas complete, when it peeled off from the surface of thewoundby itself. In our experience, the bovine amnion hasbehaved as well as the human amnion in the dry state.Douglas et al" have noted very little difference betweenheterologous andhomologous choriongrafts in the mouse.Ourexperience with the bovine amnion both in therabbitand the human subject tends to support this view. The

mainadvantageofthebovineamnionis its largesurfaceascompared with the human material. This availability oflarge surfaceovershadows the minordisadvantage of lessefficient adherence to the wound. We have found no

comparable series in literature in which thebovine amnionwas used as the solebiological dressing.

Irradiation

Irradiation has been quite effective and safe in steriliz

ing the amnion. There were no deleterious effects ofradiation 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 eliminatespore-bearing organisms, as 70% of our samples werereported to contain the nonpathogenic aerobicspore-bearing organisms. To eliminate these also, y radiation wastried, and we found that the standard dosesof y radiation

(2.5mrad)routinelyadministered forall materials isquitesafe and effective for amnion also.

Bonding

Amnion,whenspread on a plasticsheet, adheres to it asit dries. But when it isseparated from the wound surfaceby interposition of a plastic sheet between the membrane

and the wound bed, it

becomes ineffective. Thus, the



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behaviorofamniondiffersin this respectfrom apolyethylene film. The bondbetween the two is similar to and more

than that of homografts andheterografts of skin ratherthan a polyethylene film. There seems to be a biological,notmechanical, bond between thewound and theamnion.This biological bond has been considered to be a fibrinelastin biological bond.19

Advantages

Amnion is especially useful in children because themembrane,being wafer thin andlight in weight, releasesthe children from the restraining influence of bulkydressings, which may cause respiratory embarrassment whenapplied over thetrunk and abdomen.

Use of dried amnion, it is hoped, will enable efficientmanagement of burns even in rural surroundings. Theimportance of the dry membrane will be appreciated

especially in developing countries like India, where maintenance ofcostlyamnionbanks is noteasyexceptin majorcities.Furthermore,bankamnionhas alife spanofonlysixweeks, whereas thedried membrane has a life span of atleast nine months in our hands. This method of treatmentdoes not strain the finances of thepatient or thehospitalservices. The use of bovine amnion provides unlimitedsupplies of the material. The effectiveness of dried andirradiatedamnion also confirms that tissueviability is notessential.

K. L. Dutt,GMVC,ND(AR), PG (AUS),joint director and project officerofthe Indo-SwissCattle ResearchStation,Visakhapatnam, India,providedthe bovine amnion; J. Seethamma, MD, DGO, superintendent of VictoriaHospital for Women and Children, Visakhapatnam, provided the humanamnion;and P. A.Jayakar,MD,DCh,professorofmicrobiology,and B. RajaRao, MD, additional professor of microbiology, Andhar Medical College,Visakhapatnam, provided theexperimental animals.

References

1. DavisJS: Skintransplantation, with a reviewof 550 cases at the JohnHopkins Hospital. JohnsHopkinsHospRep 1910;15:307.

2. Sabella N: Use of foetal membranes in skin grafting. Med Rec NY1913;83:478.3. Johnson HL: Insulating patches and absorbable sutures made from

fetal membranes. NEngl 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 J1940;1:517-519.

5. Troensgaard-Hansen E: Amniotic grafts in chronic skin ulceration.Lancet1950;1:859-860.

6. Brown JB, Fryer MP, Rendall P, et al: Post mortem homografts asbiological dressings for extensive burns and denuded areas. Ann Surg1953;138:618-630.

7. Silvetti AN, Cotton C, ByrneRJ, et al:Preliminary studies of bovineembryoskin grafts. Transplant Bull 1957;4:25.

8. DinoBR,EufemioGG,DeVillaMS: Human amnion: The establishmentof an amnion bank and itspracticalapplications in surgery. JPhilip MedAssoc 1966;42:357-366.

9. Song IC, Bromberg BE, Mohn MP, et al: Heterografts as biologicaldressings forlarge wounds. Surgery 1966;59:576.10. Colocho G, Graham WP III, Greene AE, et al: Human amniotic

membrane as a physiologicwounddressing. ArchSurg 1974;109:370-373.11. Walker A, Cooney DR, Allen JE: Use of fresh amnion as a burn

dressing.J PediatrSurg 1977;12:391-395.12. Jenner JA: The Graftingof Preserved Fetal Membranes to DenudedSkinSurfaces. Marquette University, Milwaukee.

13. Douglas B,Conway H, Stark RB, et al: The fate ofhomologous andheterologouschorionictransplants as observedby transparent tissue cham-bertechnique in the mouse. Plast ReconstrSurg 1954;13:125-129.

14. Trelford JD, Anderson DG, Hanson FW: Considerations of theamnion as an autograft and as anallograft in sheep. J Med1972;3:231.

15. Robson MC, Krizek TJ: The effect of human amniotic membranes onthe 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 NorthAm 1970;50:1325-1335.

17. Gump FE, Kinney JM: Caloric and fluid losses through the burnwound.Surg Clin North Am 1970;50:1235-1248.

18. Pigeon J: Treatment of second-degree burns with amniotic mem-branes. Can Med Assoc J 1960;83:844.

19. BurlesonR, Eiseman B: Natureof thebondbetweenpartial-thicknessskin and woundgranulations.Surgery 1972;72:315-322.

Invited Editorial Comment

Thisreportby DrsRaoand Chandrasekharam describes the useof dried amnion from both human and bovine sources as a

biologicaldressing. Their examples of multiple kinds of wounds,both experimental and clinical, are quite impressive. They havefound that amnion has most of the properties of a biologicaldressing. However, one function that it does n ot fulfill is that of abiological-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 theeffectiveness of decreasing tissue bacterial counts is due to thevascular ingrowth into the biological-physiological membrane.

Finally, there is difficulty in using any type of biologicaldressing over a full-thickness burn. A full-thickness burn bydefinitionwill have nonviable tissue and bacteria in itsdepth. Toclosethiswith abiologicaldressingis very muchlikeputting a top

on an abscess. The authors had difficulty using it over a full-thicknesswound, and indeed this is theexperience of others whohavetried either abiological dressing or a syntheticdressingsuchasHydron WoundDressing(AbbottLaboratories, NorthChicago,111) over full-thicknessinjuries.Biologicaldressings are best usedfor partial-thickness injuries or full-thickness injuries after theescharhas been totally excised.

Despite objections to thespecificpoints mentioned, the articledescribes a wayto dryamnion and preserve it forlongperiods oftime. If one is desirous of abiological dressing to serveonly as acoverandnot function as a biological-physiologicaldressing,thesetechniques may prove to have value.

Martin C. Robson, MDChicago

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Use of Dry Human and Bovine Amnion as a Biological Dressing (2)