CURRENT DEVELOPMENTS
The amnion in surgery, past and present JOHN
D. TRELFORD,
Sacramr’nto, MARILYN
M.D.,
F.R.C.S.(C.),
F.R.C.O.G.(LOND.),
F.A.C.O.G.,
F.A.C.S.
Cabfornia TRELFORD-SAUDER,
M.D.
South Leon, Michigan, The clinical application of the fetal membranes has been under study for many years. An attempt is made to bring the reader up to date in this fascinating subject and to indicate practical employment of the membrane in modern medicine. The amnion and chorion are detailed separately where possible, particularly in the field of surgery. (AM. J. OBSTET. GYNECOL. 134:833, 1979.)
DAVID COPPERFIELD was born with a caul* and, as everyone in those days knew, that was good luck. It had been well known since early Scottish times that sailors so born carried a dry remnant of their fetal membranes, “the sely how,” to save them from drowning. In time, fetal membranes gradually lost their magic and resumed their role as nature’s refuse. Today, however, research of this surprising material in both animal and human investigations may well return it to its magical state of yore. The purpose of this paper is to report a comprehensive review of the literature, to present the current state of knowledge of the fetal membranes in terms of embryologic development, histology, and immunology, and to summarize the clinical application of this information as usecl by myself and others.1-s2 Since 1970 there have been over 20 articles on the clinical use of the fetal membranes, which is more published material than in all previous years combined. In the literature review a great deal of confusion exists, as the fetal membranes, chorion, and amnion From thP Department of Gynecologic Oncology, University of Californiu at Sacramento Medical Center. Reprint requestr: Dr. John D. Treford, Department Gynecologic Oncology, Universiq of California at Sacmmen’u Medical Center, 4301 X St., Room 207, Sacramen!o, California 95817. *The amnion delivered unruptured with the child, capping the child’s head when born. 000%9378/79/150833+
135601.3010 0
1979 The
C. V. Mosby
of
thus
Co.
were used interchangeably, particularly in the earlier literature. This fact must be taken into consideration as the chorion and amnion are distinctly separate entities biologically. In this review, quotation marks are used to identify those instances in which strict designation doe& not exist. Davis** in 1910 first reported the use of fetal membranes in skin transplantation. Stern’] and Sabella,63 working together in 1913, treated and independently reported the use of “amniotic membrane” on burned and ulcerated skin surfaces. They reasoned that the amnion was the appropriate side of the membranes to put in apposition to the skin because of its ectodermal origin. The intact membranes were applied to the wound and then covered with warm paraffin and dressings. After 48 hours the dressings were removed. The amnion was adherent to the wound. The chorion was adherent to the paraffin. They observed a lack of infection in clean or sterile wounds, a marked decrease in pain, and an increased rate of re-epithelialization of the traumatized skin surface. Sabella’j3 also used the tissues of the umbilical cord for dressings and astutely observed the unusually thick-walled veins and arteries of the cord.* Apparently their enthusiasm did not excite the medical world as no reference is found in the medical literature for the next 22 years. *Interest in these vessels for transplant grafts is now under investigation. 833
834
Trelford and Trelford-Sauder
Brindeau” in 1935 and Burger I’. ‘ji in 1937 reporte(l using “amnion” as a graft in forming an artiticial \;Igina. Although the technique of application \vas difficult to ascertain, they apparently were successful ill restoring sexual function for the patient. Vaginal scrapings taken 9 months after surgery showed normal epithelial cells that did not bear resemblance IO the membrane. It was postulated that the cells had grown in from the introitus. Burger experimented with this tissue in rabbits, dogs, and cats. He reported that no adhesions formed when the amnion was used in dura mater defects. In 1940 DeRotth2” reported its successful use in conjunctival defects. He implanted the memhranes in the peritoneum of dogs but apparentl) used both “amnion and chorion.” The adhesions that IVsulted discouraged further experiments of that naturt until a successful trial of aninion alone was done in 194X.“z III 1940 Chao and associates’g published the first paper dealing with “amnioplastin” in the prevention of meningocerebral adhesions following head injury. The “amniotic membrane” was placed in 70% alcohol and then dried in an oven. It was stored in 70% alcohol There is little doubt that the result \vas a tle.rtf parchment-like material that bears no resemblance to living grafts. Microscopically their results at 10 to 60 days after surgery showed continual dural repair with no organized adhesions or evidence of rqjection other than fibroblasts above and below the material. The “amnioplastin” itself degenerated into a mucoid substance that disappeared in 30 days. Amnioplastirl gained some popularity in peripheral nerve injuries, conjunctival grafts, and flexor and tendon repairs. Pikit?’ in 1942 used a form of “amnioplastin” in fresh trauma and reported it in the Russian literature. :\Ithough all reports were enthusiastic, mention of “amnioplastin” disappeared from the literature with no real explanation. No critical reports have been found. and if there were failures of treatment, they do not appear to have been reported. Between 1941 and 1948 Kubanyi” used live “amnion” in patients with burns and traumatic skin wounds. He reviewed Burger’s work with amnion in the abdomen. Encouraged by this, he tried it in a patient with an enterocutaneous fistula secondary to surgery for lysis of adhesions, a problem fraught with failure. After a closure of the high small bowel fistula. he then wrapped the bowel repair by the technique advocated at that time, with amnion. The operation was successful, and the patient was discharged on a regular diet 12 days after surgery. Although he advocated further intra-abdominal use of the membrane in the
treatment
ot adhesions.
hc does ilol ~~c~port :itltlitic~nal
CilseS.
In 1952 Douglas,“6, 3; working it1 (;oodpasttlre’s Iahorator);, successfully grafted human m(:mbraJlrs on the chorioallantoic membranes of chic k\. 1~ wx !hiers is e;lsiii qarated hy blunt finger tlissecticm, allo\vitig tot. total s~pardtioil of the two
cOt11polK’t1l’.
011
tk1c. 111051 oiltel‘
aspect
c)f’the
cho-
I-ion, fr-aglnctlts of trlar~t-ml decidua may he idetttified as demarkittg the line ot wparatiott bctwcen the menrbratie arid Ilrc* uteritic c;i\itl.
Histology
2, 6, 24, 31, 34, 35, 36, 44, 66, 66, 91
‘I‘he atntiioti varies iit tii5tolog! from conception to maturity. For a f’rtll, detailed descripdon one is r-tGrrctl to Hayes”‘” synopsis of’ this subject and a report by van Herendael and colleagucs.‘K Hecausc only mature rissue l‘t~mi term pregnaticies is used clinically, the description which iollows will be limited to this time period. Gonsidcration must be givrn to the $prcd and type ot preservation of the tnetnbratw before the histology is described in detail. Numerorts report5 describe rhe cl-
Volume Number
Amnion in surgery
1:bl 7
Fig. 2. Sheep experiment in which the amnion as an autograft was buried subcutaneously 5;ection was taken at the end of 9 months.
feet of changing osmolarities of fluid about the membrane that cause major artifacts. The effects of labor and removal of the membrane from the uterus must be taken into consideration. Light microscopy reveals a single layer of cuboidal epidermis-like cells on a base of scattered fibroblasts in a collagen matrix. It must be realized that throughout gestation the embryonic mesoderm gradually matures through mesenchymal cells to more mature fibroblasts as seen in adult tissues. The mature, term amnion is essentially a unilaminar epithelium overlying a layer of mesenchyme which contains large amounts of collagen, the latter cells being predominantly fibroblastic. The amnion cells have considerable variation in height, varying between columnar over the placental amnion and cuboidal or flattened cells over the extraplacental amnion. In the mature amnion layer, microvilli are numerous on the surface and la.teral aspects of the cell. These microvilli are essentially cylindrical at term, may be regular or irregular, branched, or confluent, but generally have an appearance similar to that of the brush border associated with other types of epithelia. Amorphous material can be seen on the surface of the microvilli in the term amnion and has been considered to be a mucopolysaccharidc secreted by the cells but proof is still lacking. The cytoplasmic core of the microvilli has a fine tibrillar substructure which probably has a sup-
837
as a tube.
portive function. The microvilli continue down the lateral border of the cuboidal cells to form intercellular canaliculi of the labyrinth type. Spaces between the cells of the term amnion appear to form a part of a complex and tortuous series of channels occasionally opening from the surface to the base of the cells. (It has been questioned as to whether these might be freeze fractures but no studies could be found.) The cells are united at the surface by desmosomes. Desmosomes are present at intervals along the lateral membranes of the cells. These junctional complexes add to the labyrinth arrangement of the intercellular channels. Hemidesmosomes on the basal membrane are highly developed and are most numerous in the cells of the term amniotic membrane. The basal plasma membrane in the taller cells is folded to form footlike processes although rarely present in rhe low cuboidal cells. These cell processes (pedicles), extend into the basement membrane in podocyte fashion. The basal cell processes have a hemidesmosome type of function with tonofilaments. The basement membrane substance is partly amorphous, partly microfibrillar. The ultrastructure of the nuclei of the amnion in mature gestation is different from that seen in earlier stages of gestation. The nuclei are irregular with a number of prominent but shallow indentations of the nuclear membrane. Irregular clumps of heterochro-
838
Trelford
and Treiford-Sauder
3. Sheep experiment in which an allograft arnnion was buried srlbc-utaneouaiy, Section ~a\ I,iken at day 8. Note the blood vessels immediately helo~ the mesenrh~me of thr amnion. Cotnparr to Fig. 4.
Fig.
mation are present at the periphery of the nucleus. The nucleolus is often large and homogeneous in appearance and suggesting nucleolar inactivity but there are indications that it can be stimulated. The cytoplasmic filaments are highly developed, forming a network of bundles throughout the cytoplasm, and come into close relation with the lateral membranes in the region of the desmosomes. The ribosomes are typically scattered throughout the cytoplasm. The cisternae of endoplasmic reticulum arr small but numerous. Both rough and smooth endoplasmic reticulum is present. Small, uncoated \;esicles are numerous, frequently seen near the lateral and basal membranes of the cell. The Golgi apparatus is usually supranuclear and small, surrounded by somewhat larger vesicles or smaller vacuoles. Such vesicles are scattered throughout the supranuclear cytoplasm close to and in communication with the membrane at the surface of the cell. Mitochondria are fairly numerous but do not appear to be concentrated. Paranucleal vacuoles appear to be lipid droplets. Studies suggest these to be saturated fat. There is no indication that the droplets are surrounded by a membrane. Secretor\grades may also be present. Immundogyl3,16,27,32,
30, 77, 81, 82, 3% 85
The amnion (devoid of chorion) as an autograft implanted into its own newborn infant has been shown to
“take” as a permanent graft?’ The mesenchyme cell in this circumstance must be toward the host as t.he amnion cells would not form attachments. Neovascularization did not occur. Nourishment 01 the graft appeared to be by simple diffusion (Figs. I and 2). Allografts c,f the amnion similarly implanted subcutaneously were founcl to be identical to the autografts for the tirst 14 to 17 days. Subsequentl) these grafts were reduced to hyalinized images”’ with only a mild infiltration of small. round ~~11s bv day 20 to 30 (Figs. 3 and 4). Allografts and autogratts as biological dressings for surface defects acted in an identical manner (Fig. S). “take” or “fixation” of the graft occurred Superior when the mesenchyme was placed toward the host, usually within 24 to 48 hours. If the amnion side was placed toward the host, in most instances the graft showed little fixation at the end of 72 hours. Neovascularization was not observed in either instance.“” Allograft amnion membranes placed in the pelvic cavity following exentemtive procedures were recovered at the end of 2 I days and histologically appeared to be viable.” Granulation tissue and fibroblast activity were observed to be profoundly inhibited in these cases when compared to those in which the membrane was not used. Implantation of the allograft a mnion into the intraperitoneal cavity appeared to prevent the formation of adhesions in animals in whom the cecum was
Volume Number
Amnion in surgery
134 7
Fig. 4. Sheep experiment in which an allograft amnion was buried at day 23. Note the gradual hyalinization and disappearance inflammatory response was thought to be secondary to infection phonuclear leukocytes and limited to a focal area
damaged and bacterially contaminated. The membrane gradually disintegrated with almost no host response (Figs. 6 and 7).“” Attempts to form antibodies or a cell immune response to amniotic membrane were unsuccessful, though not pursued to conclusion. The evidence presented suggests that the antigenicity of the amnion for whatever reason appears to be low, and violent host responses have not as yet been demonstrated. The chorion, when placed against the host tissue, provokes neovascularization and migration of host ceils, eventually causing a typical host-versus-graft rejection phenomenon. 2’ This tissue was recently studied and was shown to have considerable antigenicity that provoked a strong cellular response and a lesser antibody response. Surprisingly, this response was noted both as an autograft and as an allograft, being rejected by 14 days. When implanted in the maternal host, the tissue had an accelerated rejection within 72 hours, presumably due to presensitization of the maternal host by the fetus. This rejection phenomenon could be delayed by the use of high-dose progesterone. A number of investigators13, l* have located antigens on trophoblastic cells. Recently it has been reported that chorionic tissue may have three unusual antigens. Two were found in the mature placenta; one appeared relatively specific for the chorion. These observations may explain some of the incon-
839
subcutaneously. Section was taken of the membrane. The isolated
as the cells were mainly polymor-
sistencies of clinical reports. The difference in immunogenicity of these two membranes may be related to the presence of fragments of maternal decidua on the chorion. Animal experimentation suggests that the chorion alone can be responsible. If the chorion is separated from the amnion and the mesenchymal side applied to the host, a lesser reaction may be observed.
Physiology
3,4,
5, 15, 17, 29, 43, 45, 53, 54, 64-67, 72, 81
There is a paucity of information concerning the physiology of the amniotic membrane both in vitro and in vivo. Most of what is known has been studied in vitro and is pertinent for this paper as this is the clinical situation in which the membrane is used. The strength of the fetal membranes was first tested in 1866 by dropping “cannonballs” onto the membrane stretched over a ring. More recent studies have been more scientific, taking into account temperature, moisture, bacterial contamination, and other factors as well as the area sampled. MacLachlan,‘” using the intact fetal membranes, chorion and amnion, found a mean bursting pressure of 393 mm Hg with a maximum of 900 mm Hg in one case. Polishuk and associatess3 found the tensile force per unit width to range between 0.05 and 0.45 kg/cm. They reported that the smaller the fetus, the stronger the fetal membranes, reaching a critical value of Tw 0.050 l/cm, corresponding to the weight of a mature fetus not greater than 3,700 grams.
840
Trelford and Trelford-Sauder
Fig. 6. Rabbit experiment gradual disintegration.
in which the amnion 1,as left in the per-itoncal cza\-it) I’or 2 1 (Ia\ s. .Xrw
As the fetal weight increased over this value, the tensile strength decreased. No studies were found in which the amnion and chorion were studied separately. It has been postulated that the amnion is considerabl) stronger than the chorion by virtue of its increased amount of collagen. -The permeability of the fetal membranes has been studied mainly in vitro. -). 5. ” There is evidence that the
amnion probably does not operate as a simple semipermeable membrane and that the epithelial cells ma) be active in controlling ionic mowment. High levels of adenosine triphospharase in these cells suggest the possibility of active reabsorption of sodium from the fluid in the intercellular spaces. The presence of a system of intercellular channels in open communic-ation with both the amniotic cavity and the underlying
Volume Number
Amnion in surgery
134 7
Fig. 7. Human amnion used mesenchyme shows moderate
in a rotal exenteration retrieved at 21 days. Note degeneration, the amnion cell layer is still intact.
mesenchyme is probably the reason why in vitro studies have failed to (demonstrate the active physiology of this membrane. It is possible that in viva the amniotic membrane maintains a fi’qe balance between the hydrostatic and osmotic forces controlling the movement of fluid. Further studies are warranted to solve this difficult problem. In implantation studies of the amnion with the mesenchJ,me toward the host, fluid appeared within the cavity in all instances, whether as a transudate or an active excretion is unknown.R’ The presence of pedicels is highly suggestive of active transport. Recent studies have revealed fetal membrane collagens unknown in adult tissue.15 These collagens have differences in both peptide profiles and amino acid composition. The physiologic significance has not been elucidated bu,: may have a role in the unusual immunology that has been observed. Renin has been identified in the chorion but not in the amnion membrane7* Lipids identified in the amniotic membranes appear to be influenced by blood lipid content and the maternal diet.j” Increases over a normal content of lipid are mostly due to an increase in triglyceride. It has been postulated that the lipid vacuoles of the amnion cell may contribute to the lipid contents of verni.r. Lactic dehydrogenase was found to be high in “fetal membranes” and may be related to anaerobic resl3iration.“6 It is of inte.rest that amnion cells are easily grown in
that,
though
841
the
tissue culture. When “fibroblastS growth factor” of Dr. Gaspodorowicz was added to the medium, the speed of mitosis was increased by a factor of 280.2g The significance is unknown. The immunology of the amnion and chorion requires much more work. Experimental and clinical observations suggest that the chorion as an allograft or autograft is the most antigenic; the amnion as an autograft, the least, being accepted as self, and as an allograft, minimally active. “’ The chorion appears to induce neovascularization whereas this phenomenon is not seen when the amnion is used alone.“7 Induction experiments have revealed that the human amnion is capable of inducing formation of epithelial cells in the chorioallantoic membrane of fertilized chicken eggs. This method opens an avenue of research with this tissue. As yet observations have not revealed induction phenomena to occur in clinical situations6” The complexities of the amnion cell suggest that it is dynamic with multiple functions.
Clinical applications Seronegative healthy mothers in whom prolonged rupture of the membranes and infection are not a problem are chosen as donors of the membranes. Tissue taken at the time of cesarean section is ideal. Severely meconium-stained membranes are discarded. Patients with a history of hepatitis are not used as donors.
842
Trelford and Trelford-Sauder
Preparation.
10. 16, 25,41,42,
5% 60, 62, 84,67 l.he
f’et,t,L
lncI,,.
brar~x are removed by trimming them from the placenta at the time of delivery, either vaginal or by cesa~ ean section. Maternal blood, meconium. and other ww taminants are removed by washing in sterile saline until grossly clean. If just stored in sterile saline, all specimens will become contaminated in 3 days. Rohson and associatesj”. ” rinsed the membranes in a 0.0257, sodium hypochlorite solution and, when stored at 4” (: in a sterile saline solution containing penicillin, reinained sterile up to 6 weeks. Trelford and associates’” and Trelford-Sauder and ‘lrelfordx-i found that menbranes stored at 4” C; in O..iN saline to which pol!,mixin. ampicillin, gentamicin, and amphotericin B had been added were sterile at the end of’ 1 hours and remained so fi)r at least 48 hours. In these studies the constant supply of tissue from the delivery room did not necessitate longer storage. Further experiments in the stcrilization of the membranes are under study at the present time, particularly in regard to anaerobes.” Dino and associate? performed cultures to stud\ sterilization procedures of amniotic membranes. PJWervation with a I : 40 dilution of’ sodium hypochloritc revealed no positive cultures until 30 days. Similar rcsults were obtained when aqueous penicillin, .X),000 L‘. and streptomycin, 1 .O gm in 400 cc of’ normal saliw. were used. When kanamycin sulfate. 1.0 gm in 400 CC normal saline. was used, no positive cultures were f‘ound even at the end of 30 days. The bacteriostatic or bacteriocidal action of amniotic fluid has been under study. This may be of some assistance but it cannot be counted upon to guarantee sterility.” L\iophilization, formalinization. and liquid nitrogen storage have all been advocated. 19 ‘. 23. -4x.71. x7 The latrel. can maintain viability almost indefinitely as a tissue culture technique. Other than in the case of autografts where long-term viability may be desired, none ofthese methods shows advantage over the constant suppi> from the delivery room. It should be noted that if the chorion and amnion are separated and the amnion’s mesenchyme side is applied to the host tissue, then a virtually uncontaminated tissue surface is being used. The removal of the mow antigenic chorion and the placement of the uncontaminated mesenchyme toward the host has been thought to contribute to the successful use of the amnion to replace the pelvic lid after exenterations’.’ (Fig. 5).
Fetal membranes
All author-s agree that the nwtnbr:~t~~~ ICY!II(C’ l);tlrl. decrease elec.1 roivte. fiirid. dntl pi 01zili lash. .111ti 1” 01w i the raw s~~rt;tw of’the Irost. II is s~atrti I)\ IIMII~ rlr;~l 111~ final result is at least equal or supcriw 10 th,cl I)! XC”~~Ograft (por,cinc) and other surface (.o\c‘ri tlgs. I” ‘I’ Ii” ( :C)SI estimates haw varied nntong imtitutiorir biit lia\(~ bwn lower than those associated cvith xenogralts ttiw to tllc ready availabilitv of the fc.tal tnenib~~ancs Iron1 tlrc, rlc,liverv room. Sterilization of the lisbw has been \IL(((:s,~ful albeit lllan\ methods ha\e IXYAII II\c’~. Burn patients have IXTII VVY\ \Lr ainniolr surf,i(r t~w;trfl tlic* llozt fias been used as ;I permanent dressing iir (asr~ of super’ficial burns and those ~II tt hom cwh,tr. 01 iutwtloil is not a problem. In inf.wtcd case+ 01’ thaw iii \\lioln (ivbridement is newssal-\ , tr~nipor,ir\ i-c~l~l;twnieiir i\ [iiilized and the rncmbrane~ art’ c har~gt~i CVI’I \ ‘48 bout-5. Kobson and co-worker \(i’i ,tdvwatc pla( ins tttc’ c horicm against the Ii051 iri tlltw~ cawj. Ihc! 11oletl 1iw1;1\,cularization to occur II n&1- tlie4tl c ircunistai1(c5. pai titularly when lhc chorion \\‘a\ Irtt irl plx C‘ of (‘1‘ T’1 hours. It is possible that I his reactiorl of I he host m;t\ Ix beneficial uritlrr these cir~IIilIst;Ii1(‘(‘~. I IIC.thou-iori rnit! be immunologit but to date has not bwrl sho\\n IO be detrimental. N-C have 11setl amnion ,11011c \t.iih the &Ill IJl~S~llthyme tissue placed towu-cl the host and left in situ. .A clean granulation bed wady fill- alrtograt’tiug wab found in approximateI> 10 days. Pwrcntictn of’ drying of the membrane by a second membrane or. dressing was utilized. Fresh surgical wounds ma! be protect& bv amnitrtic nlembranes, 3, ni. ix. H37‘his ma\- be a skin donor siW or ;I full-thickness wound such as in the cask of radical vulvectom\, 11’~ ha\c reportcti its stwessfui list’ in lll(. l,ltter.‘“. ix, K+ Relatively firnl adheretic,? of tht, .tnmiotic ‘ membrane (nlesench!~nlr to the host) occlltwtl in -IX hours despite niovcnicrtt of lhe patient. So furthcldressings were used. Re-epithelialization o( cl~rretl under the membrane. A signihcant reduction in ho~pit;-tiization of these patients was f&rid \vhc*rl thr\- \v~‘I.(’ compared to control cases. -rhis prospfc.ti\c stud\ involved approximately 30 cases. As ment,ioned, in one case
where
necrotizing
the
entire
cellulitis,
abdominal amniotic
wail membranes
was
destro>-ed wt’fC
h)
uSed
10
protect the contents of the peritoneal cav~t! .‘!’ -l‘his technique was successfullv used to prott‘ct exposed bowel
*Recently completed studies using anaerobic and aerobic -I. cultures revealed hncornycin, neomycin, polymixin. and amphotericin B to be the superior sterilizing solution.
in surgery
in two
other
c;tses.
It must
be remembered
that
biological dressings are temporar!., until the area hc& spontaneously or until the wound is able IO be closed b) the skin of the patient.
Volume Number
Amnion in surgery
134 7
In the intraperitoneal cavity, amniotic membranes have been used to close the pelvic lid in exenterations.46 A controlled prospective series involving 24 patients animal has been reported by us. 84 As yet, sufficient work and isolated reported cases are not available to allow recommendations for the amnion to be used in intestinal surgery though the potential is definitely present. Pilot studies have been performed by our gro~p.*~ (Figs. 6 and 7.) Amnion and the umbilical cord (covered by amnion) have been used in omphalocele with lifesaving success.s”, sX In one institution in Europe this has become an established procedure and is highly recommended to others. Recent experimentation using the umbilical vessels has been reported but as yet has not become established. Amnion replacement for dura, nerve repair,
and other applications in neurosurgery in a like manner are lacking documentation of success other than in isolated instances. The use of amnion in tendon repair and synovial joints without animal experimentation cannot be recommended at this time.s2 The potential of the amniotic membrane’s application in surgery is now just becoming realized and is as wide as imagination and experimentation can make it. It may have failed to “prevent drowning” as the “sely how of yore” but is now becoming established as a successful surgical tissue of today with a bright future. This paper has been presented to bring the readers current information on this unusual membrane and stimulate future experimentation. We would like to thank Dr. Robert Cardiff, Department of Pathology, for his comments and help in editing this paper.
REFERENCES
1. Adducci, J. E.: Amnion graft, Minn. Med. 57:626, 1974. 2. Armstrong, W. D., Wilt, J. C., and Pritchard, E. T.: Vacuolation in the human amnion cell studied by time-lapse photography and electron microscopy, _ AM. 1. OBSTET. ~YNE renort on 1 a acceleration of wound healing by amnion membrane graft, Indian J. Med. Res. 62: 1342, 1974. 4. Barton, T. C., and Baker, C.: Permeability of human amnion and chorion membrane, AM. J. OBSTET. GYNECOL. 98:!562, 1967. 5. Battaglia, I’. C., et al: Glucose concentration, gradients across the maternal surface, the placenta, and the amnion of the rhesus monkey (nlacaca mulatta), AM. J. OBSTET. GYNECOL. 88:32, 1964. 6. Bergstrom. S.: Surface ultrastructure of human amnion and chorion in early pregnancy, Obstet. Gynecol. 38~513, 1971. 7. Beronilla, H. A., Jr.: Placental tissue in burns, Res. Staff Phys., September, 1973. 8. Boyd, J. D., and Hamilton, W. J.: The Human Placenta, Cambridge. 1970. W. Heffer & Sons Ltd. 9. Brindeauy A.: Creation dun vagin artificiel a l’aide des membranes ovulaires dun oeuf a terme, Gynecol. Obstet. 29~385, 1934. 10. Bromberg B. E., Song, 1. C., and Mohn, M. P.: The use of pig skin as a temporary biological dressing, Plast. Reconstr. Surg. 36:80, 1965. 11. Brown, J. B., Fryer, M. P., Randall, P., and Lu, M.: Postmortem homografts as “biological dressings” for extensive burns and denuded areas, Ann. Surg. 138:618, 1953. 12. Brown, J. B., and Fryer. M. P.: Postmortem homografts to reduce mortality in extensive burns, J. A. M. A. 165: 1163, 1954. 13. Bulienko, S. D., Fogel, P. I., Tkachishin, V. V., and Poldmar, K. S.: Antigens of the chorion, Akush. Ginekol. (Mosk.) 1:9, 1976. 14. Burger, K.: Experimental and clinical studies on transplantation of the fetal membranes, Orv. He&l. 82:800, 1938. 15. Burgeson. R. E., Adli, F. A. L., Kaitila, I. I., and Hollister,
843
16.
17.
18.
19. 20.
21. 22. 23. 24. 25.
26.
27.
D. W.: Fetal membrane collagens: Identification of two new collagen alpha chains, Proc. Natl. Acad. Sci. U. S. A. 73:2579, 1976. Burleson, R., and Riseman, B.: Mechanisms of antibacterial effect of biologic dressings, Ann. Surg. 177: 181, 1973. Carnazza, M. L. M.: The epithelium formation capacity of chorioallantoic membrane. I. Structural changes in ectodermal epithelium grafted with living tissue, Boll. Sot. Ital. Biol. Sper. 50: 1212, 1974. Carter, J.: Expression of maternal and paternal antigens on trophoblast, Nature 262:292, 1976. Chao, Y.-C., Humphreys, S., and Penfield, W.: A new method of preventing adhesions. The use of amnioplastin after craniotomy, Br. Med. J. 1:517, 1940. Chuntrasakal, C.t Clinical experiences with the use of amniotic membranes as temporary dressing in treatment of burns and.other surgical open wounds, Med. Assoc. Thailand J. 60:66, 1977. Colocho, G., Graham, W. P., Greene, A. E., Metheson, D. W., and Lynch, D.: Human amniotic membranes as a physiologic wound dressing, Arch. Surg. 109~370, 1974. Davis, J. W.: Skin transplantation with a review of 556 cases at The Johns Hopkins Hospital, Johns Hopkins Med. l. 1.5~307, 1910. DeRoith, A.: Plastic repair of conjunctival defects with fetal membranes. Arch. Onhthalmol. 23:522. 1940. DeVirgiliis, G., darinelli, S., and Remotti, d.: The chorionic membrane of the human placenta, Ann. Ostet. Ginecol. Med. Perinat. 94:239, 1973. Dino, B. R., Eufemio, G. G., and DeVilla, M. S.: Human amnion: The establishment of an amnion bank and its practical applications in surgery, J. Philippine Med. Assoc. 42:357, 1966. Douglas, B.: Homografts of fetal membranes as a covering for large wounds-especially those from burns. An experimental and clinical study, Tenn. State Med. J. 45~230. 1952. Douglas, B., Conway, H., Stark, R. B., Joslin, D., and Nieto-Cano, G.: The fate of homologous and heterolo gous chorionic transplants as observed by the transparent tissue chamber technique in the mouse, Plast. Reconstr. Surg. 13:125, 1954.
844
2x.
29. 30.
31
:42.
33.
94.
35. 36.
37. 3x.
YY.
40.
41.
42. 43.
44.
46.
47. 48. 49. 50. 51.
Trelford
and
Trelford-Sauder
Eltlad, A.. Stark, M., Anais, D., Golan, j., and BellHnr, N.: .4mniotic membranes as a biological dressit1g. S. Afr. Med. J. 51:272. 1977. (GaspodorawicT, D.: Secret to lesion repair? Hosp. ‘l-rib.. p. I. October 3, 1977. (;harib, M.: Repair of prenatally ruptured onlphaloccle and the pardumbilical abdominal wall defect with the ir1fant’s own fetal membranes, Munch. Med. Wochensch1. 117:15s5 , , 19x. Hamilton, W. J., Boyd, J. I)., and Mossman, H. W .: Human Embryology. Baltimore, 1962, The Williams k N’ilkins (Zo. llasek, M.. Haskova, V., Lengerova, A.. and Vojtisko\a, M.: Mother-foetus immunological relationship as at1 exceptional homograft model, in Wolstenholme, G. E. W., and Cameron, M. P., editors: Ciba Foundation Symposiu m on Tr~tnsplantarion, Boston, 1962, Little, Brow11 & c:o.. p. 118. Her-nandez-Verdun, I)., and Legrand, C.: In vitro stud\ of chorionic and ectoplacental rrophoblast differentiation in the mouse, J. EmbryoI. Exp. Morphol. 34:633, 1975. Hoyes, A. D.: Fine structure of’ human amniotic epithclium in rat-l! pregnancy, Br. J. Obster. Gynaecol. 75:Y49. 196X. Ho\ea, A. I).: Structure and function of the amniott. Ohster. Gynecol. Annu. 4: 1. 197.5. Jenkinson, E. J., and Billington, W. D.: Studies on the ‘immunobiology of mouse fetal membranes: The effect of cell-mediated immunity on yolk sac cells in Gtn~. ,J. Rcprod. Frrtil. 41:4OY. 1974. Karol?. B.: Eredeti Kozlemenyek. Orv. Hetil. 33:HOO. I Y3X. Kim. C. K., and Benirschke, K.: Autoradiographic stud! of the “X” cells in the hutnan placenta, Abt. J. OBSTET. GYNECOL.. 109:96, 1971. Klen, R., and Skalska. H.: A comparison of dermoepidermal and chorion-amniotic grafts in the treatment of burns, Acta Chir. Plast. (Prague) l&225, 1976. Korhary. P.: Total glossectomy and repair with *tic membrane (preliminary observations), Indian I%& Assoc.. J. 62:87, 1974. Krizck, T. J.: Biologic dressings, i?t Symposiwrn OR Basic Science in Plastic Surgery. St. Louis, 1976, The C. V. Mosby Co., p. 61. Kubanyi, A.: Trapianto d’atnnion sterile ot&eiwto da1 teglie cesareo, Ann. Ital. Chir. 25: IO, 1948. Lapan- B., and Friedman. M. M.: Tissue ads in gestation: Comparative activities in the p membranes, AM.J. OBSTET. GYNECOL. Ludwig, H., Metzger, H., Korte, M., a internal surface of the amniotic epit naekol. 217: 141, lY7-1. MacLachlan, T. B.: A method for &se itlvortigation of the swength of the fetal membraaerr, AM. J. OBST~T. GYNLCOL. 91:309, 1965. Massec, J. S.. Symmonds, R. E., l%z&&y, M. B., and Gallenback, G. A.: Use of fetal as replacement for pelvic peritoneum after aenteration in the dog, Gynecol. Obstet. 13:407 Ninman, C., and Shoemaker, P.: amniotic membt-anes for hrtrtrs, .4mer. J. Nut-s. 1975. Norca, E., Hirshowitz, B., Karev, A., et al.: Lyophilized amnion in burris and skin loss, N;rrc&llh 16~265, 1975. O’Neill, J. A., Jr.: Temporary -@I croverage with nllografts, Mod. Med., p. 163, A e burns with atnPigeon. J.: Treatment o nioric membranes, Can. Pikin. K. 1.: Therapy of of c-ltcmically treated tisswrs, !&x. Med. 6(9): 15. 1942.
52. 53.
54.
.%.
56. 57.
Pinkerton, M. (:.: Amnioplastitt 101 xll~cr ml tiigi1,11 flexor tendons, I.ancet 1:70, l!bt2. Polishuk, W. %.. Kohanc. S.. and Hatla~~, ;\.: lFt*r,tl \\(~tgltt and membrane ret1sik str~~q$t. .2~t. J, 01s\,t 1,I ( ;T\I c 01. 88:247. IS64 Polishuk. t\;. /.., Boxet J,. .+ttd (&111/ttirtl. I
The amnion in surgery, past and present JOHN
D. TRELFORD,
Sacramr’nto, MARILYN
M.D.,
F.R.C.S.(C.),
F.R.C.O.G.(LOND.),
F.A.C.O.G.,
F.A.C.S.
Cabfornia TRELFORD-SAUDER,
M.D.
South Leon, Michigan, The clinical application of the fetal membranes has been under study for many years. An attempt is made to bring the reader up to date in this fascinating subject and to indicate practical employment of the membrane in modern medicine. The amnion and chorion are detailed separately where possible, particularly in the field of surgery. (AM. J. OBSTET. GYNECOL. 134:833, 1979.)
DAVID COPPERFIELD was born with a caul* and, as everyone in those days knew, that was good luck. It had been well known since early Scottish times that sailors so born carried a dry remnant of their fetal membranes, “the sely how,” to save them from drowning. In time, fetal membranes gradually lost their magic and resumed their role as nature’s refuse. Today, however, research of this surprising material in both animal and human investigations may well return it to its magical state of yore. The purpose of this paper is to report a comprehensive review of the literature, to present the current state of knowledge of the fetal membranes in terms of embryologic development, histology, and immunology, and to summarize the clinical application of this information as usecl by myself and others.1-s2 Since 1970 there have been over 20 articles on the clinical use of the fetal membranes, which is more published material than in all previous years combined. In the literature review a great deal of confusion exists, as the fetal membranes, chorion, and amnion From thP Department of Gynecologic Oncology, University of Californiu at Sacramento Medical Center. Reprint requestr: Dr. John D. Treford, Department Gynecologic Oncology, Universiq of California at Sacmmen’u Medical Center, 4301 X St., Room 207, Sacramen!o, California 95817. *The amnion delivered unruptured with the child, capping the child’s head when born. 000%9378/79/150833+
135601.3010 0
1979 The
C. V. Mosby
of
thus
Co.
were used interchangeably, particularly in the earlier literature. This fact must be taken into consideration as the chorion and amnion are distinctly separate entities biologically. In this review, quotation marks are used to identify those instances in which strict designation doe& not exist. Davis** in 1910 first reported the use of fetal membranes in skin transplantation. Stern’] and Sabella,63 working together in 1913, treated and independently reported the use of “amniotic membrane” on burned and ulcerated skin surfaces. They reasoned that the amnion was the appropriate side of the membranes to put in apposition to the skin because of its ectodermal origin. The intact membranes were applied to the wound and then covered with warm paraffin and dressings. After 48 hours the dressings were removed. The amnion was adherent to the wound. The chorion was adherent to the paraffin. They observed a lack of infection in clean or sterile wounds, a marked decrease in pain, and an increased rate of re-epithelialization of the traumatized skin surface. Sabella’j3 also used the tissues of the umbilical cord for dressings and astutely observed the unusually thick-walled veins and arteries of the cord.* Apparently their enthusiasm did not excite the medical world as no reference is found in the medical literature for the next 22 years. *Interest in these vessels for transplant grafts is now under investigation. 833
834
Trelford and Trelford-Sauder
Brindeau” in 1935 and Burger I’. ‘ji in 1937 reporte(l using “amnion” as a graft in forming an artiticial \;Igina. Although the technique of application \vas difficult to ascertain, they apparently were successful ill restoring sexual function for the patient. Vaginal scrapings taken 9 months after surgery showed normal epithelial cells that did not bear resemblance IO the membrane. It was postulated that the cells had grown in from the introitus. Burger experimented with this tissue in rabbits, dogs, and cats. He reported that no adhesions formed when the amnion was used in dura mater defects. In 1940 DeRotth2” reported its successful use in conjunctival defects. He implanted the memhranes in the peritoneum of dogs but apparentl) used both “amnion and chorion.” The adhesions that IVsulted discouraged further experiments of that naturt until a successful trial of aninion alone was done in 194X.“z III 1940 Chao and associates’g published the first paper dealing with “amnioplastin” in the prevention of meningocerebral adhesions following head injury. The “amniotic membrane” was placed in 70% alcohol and then dried in an oven. It was stored in 70% alcohol There is little doubt that the result \vas a tle.rtf parchment-like material that bears no resemblance to living grafts. Microscopically their results at 10 to 60 days after surgery showed continual dural repair with no organized adhesions or evidence of rqjection other than fibroblasts above and below the material. The “amnioplastin” itself degenerated into a mucoid substance that disappeared in 30 days. Amnioplastirl gained some popularity in peripheral nerve injuries, conjunctival grafts, and flexor and tendon repairs. Pikit?’ in 1942 used a form of “amnioplastin” in fresh trauma and reported it in the Russian literature. :\Ithough all reports were enthusiastic, mention of “amnioplastin” disappeared from the literature with no real explanation. No critical reports have been found. and if there were failures of treatment, they do not appear to have been reported. Between 1941 and 1948 Kubanyi” used live “amnion” in patients with burns and traumatic skin wounds. He reviewed Burger’s work with amnion in the abdomen. Encouraged by this, he tried it in a patient with an enterocutaneous fistula secondary to surgery for lysis of adhesions, a problem fraught with failure. After a closure of the high small bowel fistula. he then wrapped the bowel repair by the technique advocated at that time, with amnion. The operation was successful, and the patient was discharged on a regular diet 12 days after surgery. Although he advocated further intra-abdominal use of the membrane in the
treatment
ot adhesions.
hc does ilol ~~c~port :itltlitic~nal
CilseS.
In 1952 Douglas,“6, 3; working it1 (;oodpasttlre’s Iahorator);, successfully grafted human m(:mbraJlrs on the chorioallantoic membranes of chic k\. 1~ wx !hiers is e;lsiii qarated hy blunt finger tlissecticm, allo\vitig tot. total s~pardtioil of the two
cOt11polK’t1l’.
011
tk1c. 111051 oiltel‘
aspect
c)f’the
cho-
I-ion, fr-aglnctlts of trlar~t-ml decidua may he idetttified as demarkittg the line ot wparatiott bctwcen the menrbratie arid Ilrc* uteritic c;i\itl.
Histology
2, 6, 24, 31, 34, 35, 36, 44, 66, 66, 91
‘I‘he atntiioti varies iit tii5tolog! from conception to maturity. For a f’rtll, detailed descripdon one is r-tGrrctl to Hayes”‘” synopsis of’ this subject and a report by van Herendael and colleagucs.‘K Hecausc only mature rissue l‘t~mi term pregnaticies is used clinically, the description which iollows will be limited to this time period. Gonsidcration must be givrn to the $prcd and type ot preservation of the tnetnbratw before the histology is described in detail. Numerorts report5 describe rhe cl-
Volume Number
Amnion in surgery
1:bl 7
Fig. 2. Sheep experiment in which the amnion as an autograft was buried subcutaneously 5;ection was taken at the end of 9 months.
feet of changing osmolarities of fluid about the membrane that cause major artifacts. The effects of labor and removal of the membrane from the uterus must be taken into consideration. Light microscopy reveals a single layer of cuboidal epidermis-like cells on a base of scattered fibroblasts in a collagen matrix. It must be realized that throughout gestation the embryonic mesoderm gradually matures through mesenchymal cells to more mature fibroblasts as seen in adult tissues. The mature, term amnion is essentially a unilaminar epithelium overlying a layer of mesenchyme which contains large amounts of collagen, the latter cells being predominantly fibroblastic. The amnion cells have considerable variation in height, varying between columnar over the placental amnion and cuboidal or flattened cells over the extraplacental amnion. In the mature amnion layer, microvilli are numerous on the surface and la.teral aspects of the cell. These microvilli are essentially cylindrical at term, may be regular or irregular, branched, or confluent, but generally have an appearance similar to that of the brush border associated with other types of epithelia. Amorphous material can be seen on the surface of the microvilli in the term amnion and has been considered to be a mucopolysaccharidc secreted by the cells but proof is still lacking. The cytoplasmic core of the microvilli has a fine tibrillar substructure which probably has a sup-
837
as a tube.
portive function. The microvilli continue down the lateral border of the cuboidal cells to form intercellular canaliculi of the labyrinth type. Spaces between the cells of the term amnion appear to form a part of a complex and tortuous series of channels occasionally opening from the surface to the base of the cells. (It has been questioned as to whether these might be freeze fractures but no studies could be found.) The cells are united at the surface by desmosomes. Desmosomes are present at intervals along the lateral membranes of the cells. These junctional complexes add to the labyrinth arrangement of the intercellular channels. Hemidesmosomes on the basal membrane are highly developed and are most numerous in the cells of the term amniotic membrane. The basal plasma membrane in the taller cells is folded to form footlike processes although rarely present in rhe low cuboidal cells. These cell processes (pedicles), extend into the basement membrane in podocyte fashion. The basal cell processes have a hemidesmosome type of function with tonofilaments. The basement membrane substance is partly amorphous, partly microfibrillar. The ultrastructure of the nuclei of the amnion in mature gestation is different from that seen in earlier stages of gestation. The nuclei are irregular with a number of prominent but shallow indentations of the nuclear membrane. Irregular clumps of heterochro-
838
Trelford
and Treiford-Sauder
3. Sheep experiment in which an allograft arnnion was buried srlbc-utaneouaiy, Section ~a\ I,iken at day 8. Note the blood vessels immediately helo~ the mesenrh~me of thr amnion. Cotnparr to Fig. 4.
Fig.
mation are present at the periphery of the nucleus. The nucleolus is often large and homogeneous in appearance and suggesting nucleolar inactivity but there are indications that it can be stimulated. The cytoplasmic filaments are highly developed, forming a network of bundles throughout the cytoplasm, and come into close relation with the lateral membranes in the region of the desmosomes. The ribosomes are typically scattered throughout the cytoplasm. The cisternae of endoplasmic reticulum arr small but numerous. Both rough and smooth endoplasmic reticulum is present. Small, uncoated \;esicles are numerous, frequently seen near the lateral and basal membranes of the cell. The Golgi apparatus is usually supranuclear and small, surrounded by somewhat larger vesicles or smaller vacuoles. Such vesicles are scattered throughout the supranuclear cytoplasm close to and in communication with the membrane at the surface of the cell. Mitochondria are fairly numerous but do not appear to be concentrated. Paranucleal vacuoles appear to be lipid droplets. Studies suggest these to be saturated fat. There is no indication that the droplets are surrounded by a membrane. Secretor\grades may also be present. Immundogyl3,16,27,32,
30, 77, 81, 82, 3% 85
The amnion (devoid of chorion) as an autograft implanted into its own newborn infant has been shown to
“take” as a permanent graft?’ The mesenchyme cell in this circumstance must be toward the host as t.he amnion cells would not form attachments. Neovascularization did not occur. Nourishment 01 the graft appeared to be by simple diffusion (Figs. I and 2). Allografts c,f the amnion similarly implanted subcutaneously were founcl to be identical to the autografts for the tirst 14 to 17 days. Subsequentl) these grafts were reduced to hyalinized images”’ with only a mild infiltration of small. round ~~11s bv day 20 to 30 (Figs. 3 and 4). Allografts and autogratts as biological dressings for surface defects acted in an identical manner (Fig. S). “take” or “fixation” of the graft occurred Superior when the mesenchyme was placed toward the host, usually within 24 to 48 hours. If the amnion side was placed toward the host, in most instances the graft showed little fixation at the end of 72 hours. Neovascularization was not observed in either instance.“” Allograft amnion membranes placed in the pelvic cavity following exentemtive procedures were recovered at the end of 2 I days and histologically appeared to be viable.” Granulation tissue and fibroblast activity were observed to be profoundly inhibited in these cases when compared to those in which the membrane was not used. Implantation of the allograft a mnion into the intraperitoneal cavity appeared to prevent the formation of adhesions in animals in whom the cecum was
Volume Number
Amnion in surgery
134 7
Fig. 4. Sheep experiment in which an allograft amnion was buried at day 23. Note the gradual hyalinization and disappearance inflammatory response was thought to be secondary to infection phonuclear leukocytes and limited to a focal area
damaged and bacterially contaminated. The membrane gradually disintegrated with almost no host response (Figs. 6 and 7).“” Attempts to form antibodies or a cell immune response to amniotic membrane were unsuccessful, though not pursued to conclusion. The evidence presented suggests that the antigenicity of the amnion for whatever reason appears to be low, and violent host responses have not as yet been demonstrated. The chorion, when placed against the host tissue, provokes neovascularization and migration of host ceils, eventually causing a typical host-versus-graft rejection phenomenon. 2’ This tissue was recently studied and was shown to have considerable antigenicity that provoked a strong cellular response and a lesser antibody response. Surprisingly, this response was noted both as an autograft and as an allograft, being rejected by 14 days. When implanted in the maternal host, the tissue had an accelerated rejection within 72 hours, presumably due to presensitization of the maternal host by the fetus. This rejection phenomenon could be delayed by the use of high-dose progesterone. A number of investigators13, l* have located antigens on trophoblastic cells. Recently it has been reported that chorionic tissue may have three unusual antigens. Two were found in the mature placenta; one appeared relatively specific for the chorion. These observations may explain some of the incon-
839
subcutaneously. Section was taken of the membrane. The isolated
as the cells were mainly polymor-
sistencies of clinical reports. The difference in immunogenicity of these two membranes may be related to the presence of fragments of maternal decidua on the chorion. Animal experimentation suggests that the chorion alone can be responsible. If the chorion is separated from the amnion and the mesenchymal side applied to the host, a lesser reaction may be observed.
Physiology
3,4,
5, 15, 17, 29, 43, 45, 53, 54, 64-67, 72, 81
There is a paucity of information concerning the physiology of the amniotic membrane both in vitro and in vivo. Most of what is known has been studied in vitro and is pertinent for this paper as this is the clinical situation in which the membrane is used. The strength of the fetal membranes was first tested in 1866 by dropping “cannonballs” onto the membrane stretched over a ring. More recent studies have been more scientific, taking into account temperature, moisture, bacterial contamination, and other factors as well as the area sampled. MacLachlan,‘” using the intact fetal membranes, chorion and amnion, found a mean bursting pressure of 393 mm Hg with a maximum of 900 mm Hg in one case. Polishuk and associatess3 found the tensile force per unit width to range between 0.05 and 0.45 kg/cm. They reported that the smaller the fetus, the stronger the fetal membranes, reaching a critical value of Tw 0.050 l/cm, corresponding to the weight of a mature fetus not greater than 3,700 grams.
840
Trelford and Trelford-Sauder
Fig. 6. Rabbit experiment gradual disintegration.
in which the amnion 1,as left in the per-itoncal cza\-it) I’or 2 1 (Ia\ s. .Xrw
As the fetal weight increased over this value, the tensile strength decreased. No studies were found in which the amnion and chorion were studied separately. It has been postulated that the amnion is considerabl) stronger than the chorion by virtue of its increased amount of collagen. -The permeability of the fetal membranes has been studied mainly in vitro. -). 5. ” There is evidence that the
amnion probably does not operate as a simple semipermeable membrane and that the epithelial cells ma) be active in controlling ionic mowment. High levels of adenosine triphospharase in these cells suggest the possibility of active reabsorption of sodium from the fluid in the intercellular spaces. The presence of a system of intercellular channels in open communic-ation with both the amniotic cavity and the underlying
Volume Number
Amnion in surgery
134 7
Fig. 7. Human amnion used mesenchyme shows moderate
in a rotal exenteration retrieved at 21 days. Note degeneration, the amnion cell layer is still intact.
mesenchyme is probably the reason why in vitro studies have failed to (demonstrate the active physiology of this membrane. It is possible that in viva the amniotic membrane maintains a fi’qe balance between the hydrostatic and osmotic forces controlling the movement of fluid. Further studies are warranted to solve this difficult problem. In implantation studies of the amnion with the mesenchJ,me toward the host, fluid appeared within the cavity in all instances, whether as a transudate or an active excretion is unknown.R’ The presence of pedicels is highly suggestive of active transport. Recent studies have revealed fetal membrane collagens unknown in adult tissue.15 These collagens have differences in both peptide profiles and amino acid composition. The physiologic significance has not been elucidated bu,: may have a role in the unusual immunology that has been observed. Renin has been identified in the chorion but not in the amnion membrane7* Lipids identified in the amniotic membranes appear to be influenced by blood lipid content and the maternal diet.j” Increases over a normal content of lipid are mostly due to an increase in triglyceride. It has been postulated that the lipid vacuoles of the amnion cell may contribute to the lipid contents of verni.r. Lactic dehydrogenase was found to be high in “fetal membranes” and may be related to anaerobic resl3iration.“6 It is of inte.rest that amnion cells are easily grown in
that,
though
841
the
tissue culture. When “fibroblastS growth factor” of Dr. Gaspodorowicz was added to the medium, the speed of mitosis was increased by a factor of 280.2g The significance is unknown. The immunology of the amnion and chorion requires much more work. Experimental and clinical observations suggest that the chorion as an allograft or autograft is the most antigenic; the amnion as an autograft, the least, being accepted as self, and as an allograft, minimally active. “’ The chorion appears to induce neovascularization whereas this phenomenon is not seen when the amnion is used alone.“7 Induction experiments have revealed that the human amnion is capable of inducing formation of epithelial cells in the chorioallantoic membrane of fertilized chicken eggs. This method opens an avenue of research with this tissue. As yet observations have not revealed induction phenomena to occur in clinical situations6” The complexities of the amnion cell suggest that it is dynamic with multiple functions.
Clinical applications Seronegative healthy mothers in whom prolonged rupture of the membranes and infection are not a problem are chosen as donors of the membranes. Tissue taken at the time of cesarean section is ideal. Severely meconium-stained membranes are discarded. Patients with a history of hepatitis are not used as donors.
842
Trelford and Trelford-Sauder
Preparation.
10. 16, 25,41,42,
5% 60, 62, 84,67 l.he
f’et,t,L
lncI,,.
brar~x are removed by trimming them from the placenta at the time of delivery, either vaginal or by cesa~ ean section. Maternal blood, meconium. and other ww taminants are removed by washing in sterile saline until grossly clean. If just stored in sterile saline, all specimens will become contaminated in 3 days. Rohson and associatesj”. ” rinsed the membranes in a 0.0257, sodium hypochlorite solution and, when stored at 4” (: in a sterile saline solution containing penicillin, reinained sterile up to 6 weeks. Trelford and associates’” and Trelford-Sauder and ‘lrelfordx-i found that menbranes stored at 4” C; in O..iN saline to which pol!,mixin. ampicillin, gentamicin, and amphotericin B had been added were sterile at the end of’ 1 hours and remained so fi)r at least 48 hours. In these studies the constant supply of tissue from the delivery room did not necessitate longer storage. Further experiments in the stcrilization of the membranes are under study at the present time, particularly in regard to anaerobes.” Dino and associate? performed cultures to stud\ sterilization procedures of amniotic membranes. PJWervation with a I : 40 dilution of’ sodium hypochloritc revealed no positive cultures until 30 days. Similar rcsults were obtained when aqueous penicillin, .X),000 L‘. and streptomycin, 1 .O gm in 400 cc of’ normal saliw. were used. When kanamycin sulfate. 1.0 gm in 400 CC normal saline. was used, no positive cultures were f‘ound even at the end of 30 days. The bacteriostatic or bacteriocidal action of amniotic fluid has been under study. This may be of some assistance but it cannot be counted upon to guarantee sterility.” L\iophilization, formalinization. and liquid nitrogen storage have all been advocated. 19 ‘. 23. -4x.71. x7 The latrel. can maintain viability almost indefinitely as a tissue culture technique. Other than in the case of autografts where long-term viability may be desired, none ofthese methods shows advantage over the constant suppi> from the delivery room. It should be noted that if the chorion and amnion are separated and the amnion’s mesenchyme side is applied to the host tissue, then a virtually uncontaminated tissue surface is being used. The removal of the mow antigenic chorion and the placement of the uncontaminated mesenchyme toward the host has been thought to contribute to the successful use of the amnion to replace the pelvic lid after exenterations’.’ (Fig. 5).
Fetal membranes
All author-s agree that the nwtnbr:~t~~~ ICY!II(C’ l);tlrl. decrease elec.1 roivte. fiirid. dntl pi 01zili lash. .111ti 1” 01w i the raw s~~rt;tw of’the Irost. II is s~atrti I)\ IIMII~ rlr;~l 111~ final result is at least equal or supcriw 10 th,cl I)! XC”~~Ograft (por,cinc) and other surface (.o\c‘ri tlgs. I” ‘I’ Ii” ( :C)SI estimates haw varied nntong imtitutiorir biit lia\(~ bwn lower than those associated cvith xenogralts ttiw to tllc ready availabilitv of the fc.tal tnenib~~ancs Iron1 tlrc, rlc,liverv room. Sterilization of the lisbw has been \IL(((:s,~ful albeit lllan\ methods ha\e IXYAII II\c’~. Burn patients have IXTII VVY\ \Lr ainniolr surf,i(r t~w;trfl tlic* llozt fias been used as ;I permanent dressing iir (asr~ of super’ficial burns and those ~II tt hom cwh,tr. 01 iutwtloil is not a problem. In inf.wtcd case+ 01’ thaw iii \\lioln (ivbridement is newssal-\ , tr~nipor,ir\ i-c~l~l;twnieiir i\ [iiilized and the rncmbrane~ art’ c har~gt~i CVI’I \ ‘48 bout-5. Kobson and co-worker \(i’i ,tdvwatc pla( ins tttc’ c horicm against the Ii051 iri tlltw~ cawj. Ihc! 11oletl 1iw1;1\,cularization to occur II n&1- tlie4tl c ircunistai1(c5. pai titularly when lhc chorion \\‘a\ Irtt irl plx C‘ of (‘1‘ T’1 hours. It is possible that I his reactiorl of I he host m;t\ Ix beneficial uritlrr these cir~IIilIst;Ii1(‘(‘~. I IIC.thou-iori rnit! be immunologit but to date has not bwrl sho\\n IO be detrimental. N-C have 11setl amnion ,11011c \t.iih the &Ill IJl~S~llthyme tissue placed towu-cl the host and left in situ. .A clean granulation bed wady fill- alrtograt’tiug wab found in approximateI> 10 days. Pwrcntictn of’ drying of the membrane by a second membrane or. dressing was utilized. Fresh surgical wounds ma! be protect& bv amnitrtic nlembranes, 3, ni. ix. H37‘his ma\- be a skin donor siW or ;I full-thickness wound such as in the cask of radical vulvectom\, 11’~ ha\c reportcti its stwessfui list’ in lll(. l,ltter.‘“. ix, K+ Relatively firnl adheretic,? of tht, .tnmiotic ‘ membrane (nlesench!~nlr to the host) occlltwtl in -IX hours despite niovcnicrtt of lhe patient. So furthcldressings were used. Re-epithelialization o( cl~rretl under the membrane. A signihcant reduction in ho~pit;-tiization of these patients was f&rid \vhc*rl thr\- \v~‘I.(’ compared to control cases. -rhis prospfc.ti\c stud\ involved approximately 30 cases. As ment,ioned, in one case
where
necrotizing
the
entire
cellulitis,
abdominal amniotic
wail membranes
was
destro>-ed wt’fC
h)
uSed
10
protect the contents of the peritoneal cav~t! .‘!’ -l‘his technique was successfullv used to prott‘ct exposed bowel
*Recently completed studies using anaerobic and aerobic -I. cultures revealed hncornycin, neomycin, polymixin. and amphotericin B to be the superior sterilizing solution.
in surgery
in two
other
c;tses.
It must
be remembered
that
biological dressings are temporar!., until the area hc& spontaneously or until the wound is able IO be closed b) the skin of the patient.
Volume Number
Amnion in surgery
134 7
In the intraperitoneal cavity, amniotic membranes have been used to close the pelvic lid in exenterations.46 A controlled prospective series involving 24 patients animal has been reported by us. 84 As yet, sufficient work and isolated reported cases are not available to allow recommendations for the amnion to be used in intestinal surgery though the potential is definitely present. Pilot studies have been performed by our gro~p.*~ (Figs. 6 and 7.) Amnion and the umbilical cord (covered by amnion) have been used in omphalocele with lifesaving success.s”, sX In one institution in Europe this has become an established procedure and is highly recommended to others. Recent experimentation using the umbilical vessels has been reported but as yet has not become established. Amnion replacement for dura, nerve repair,
and other applications in neurosurgery in a like manner are lacking documentation of success other than in isolated instances. The use of amnion in tendon repair and synovial joints without animal experimentation cannot be recommended at this time.s2 The potential of the amniotic membrane’s application in surgery is now just becoming realized and is as wide as imagination and experimentation can make it. It may have failed to “prevent drowning” as the “sely how of yore” but is now becoming established as a successful surgical tissue of today with a bright future. This paper has been presented to bring the readers current information on this unusual membrane and stimulate future experimentation. We would like to thank Dr. Robert Cardiff, Department of Pathology, for his comments and help in editing this paper.
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