1156

HUMAN AMNION AS AN ADJUNCT IN WOUND Patients and Methods

HEALING membranes were collected and main-
Extra-embryonic
as described elsewhere.12,13 The amnion
tained in tissue culture
W. PAGE FAULK RICHARD MATTHEWS epithelium and its basement membrane were separated by
PETER J. STEVENS JOHN P. BENNETT blunt dissection from the underlying amniotic mesenchyme
HUGO BURGOS BAE-LI HSI and chorion immediately before application to leg ulcers.
These tissues demonstrated good viability for up to 3 weeks in
Blond McIndoe Centre for Transplantation Biology, Queen
culture.13
Victoria Hospital, East Grinstead, Sussex RH19 3DZ
Fifteen with chronic leg ulcers (of up to 40 years
patients12
duration)which were refractory to conservative and surgical
Summary Biopsy specimens from the beds of leg methods of treatment, including autografting in eleven cases,
ulcers of fifteen patients were obtained were admitted and kept on strict bed rest throughout treat-
before and after the application for 5 days of cultured ment. Their surgical management is reported in an accom-
human amnion. After amnion application there was con- panying paper.n
siderable granulation tissue in the ulcer bed and micro- Before and 5 days after amnion application two adjacent
scopical evidence of thinned connective tissues, vessel biopsy specimens were obtained aseptically from the ulcer bed.
One was placed in 10% buffered formalin for haematoxylin and
development, more compact resolution of vascular base- eosin (H and E) and reticulin stains. 14 The other was snap
ment membranes, and many more factor VIII granules
frozen in liquid-nitrogen-cooled isopentane, and cryostat sec-
within endothelial cells. These findings suggest the pre-
tions were prepared on microscope slides without chemical
sence of angiogenic factors in human amnion and this
fixation and processed for immunohistology .15 Rabbit mono-
could explain the hitherto unexplained success of amni- specific antiserum to human factor VIII was obtained from the
otic membranes in surgical practice. Netherlands Red Cross Blood Transfusion Service and a fluor-
escein-isothiocyanate (FITC) conjugate of sheep anti-rabbit
Introduction immunoglobulin (Ig) was obtained from Burroughs Wellcome,
Kent. Fluorescence microscopy16 was performed with a Zeiss
Wourrn healing is affected adversely by poor general
Universal microscope fitted with an FITC interference primary
health and several local factors such as inadequate
filter, dark ground condensor, and a 520 nm barrier filter.
blood-supply and chronic infections. If direct closure of
the wound is impracticable, a healthy granulating Results
wound bed which will close either by marginal epithelia-
lisation or by autografting is desirable. The plethora of Gross appearance.-In most patients a clean, red,
delicate layer of granulation tissue had formed through-
creams, powders, solutions, and dressings used to pro-
out the wound bed after 5 days of amnion application.
mote wound healing indicates the incomplete state of
Some had a less exuberant response, but all wound beds
knowledge on this subject. Human amnion has been
sporadically used, since at least 1910, to promote the produced punctate bleeding after moderate to firm rub-
formation of granulation tissues,1-4 and lately as a biolo- bing of the surface with a gauze swab. When the superfi-
cial granulation tissue was removed the underlying sur-
gical dressing for open wounds,s-’ including burns8-11 face also bled freely.
and chronic ulceration of the legs.12 However, the vascu-
lar events which are initiated by this therapy have not
been investigated. We present histopathological and im- Histopathology.-H and E stains of biopsy specimens
munohistological evidence for the development of pro- from the ulcer bed before amnion application showed
fuse granulation tissue in the ulcer bed after amnion few small vessels (fig. 1A), but biopsy specimens taken
application to leg ulcers. after application of amnion showed many capillaries
(fig. 1B). Reticulin stains of ulcer-bed tissues before
amnion application contained dense connective tissue
that tended to isolate groups of thick-walled vessels with
disordered endothelium; many of the vessels appeared
not to be patent (fig. 2A). After 5 days of amnion appli-
2. Sabella N. Use of fetal membranes in skin grafting. Med Records NY 1913;
83:478-80. cation the connective tissue fibres were more delicate
3. Troensegaard-Hansen E. Amniotic grafts in chronic skin ulceration. Lancet and the vessels seemed to be more numerous, evenly dis-
1950; i: 859-60.
4. Robson MC, Krizek TJ. Amniotic membranes as a temporary wound dress- persed, thin-walled, and patent, and the endothelium
ing. Surg Gynæcol Obstet 1973; 136: 904-06. was often more clearly defined (fig. 2B). Biopsy speci-
5. Gruss JS, Jirsch DW. Human amniotic membrane: a versatile wound dress-
mens taken before amnion application showed promi-
ing. J Can Med Assoc 1978; 118: 1237-46.
6. Bose B. Burn wound dressing with human amniotic membrane. Ann R Coil nent infiltrates of polymorphonuclear leucocytes and
SurgEng 1979; 61: 444-47. mononuclear cells, often with the morphological charac-
7. Kinmonth JB, Rob CG, Simeone FA, eds. Ulcers of the legs and feet. In: Vas-
cular surgery. London: Edward Arnold 1962: 330-36. teristics of plasma-cells.
8. Summers FH, McLaughlin CR. Emulsifying eusol/liquid paraffin. Lancet
1968; ii: 1299.
9. Bourne G. Human amnion and chorion. London: Lloyd-Luke, 1962: 10.
10. Colocho G, Graham III WP, Green AE, Matheson DW, Lynch D. Human Immunohistology.-Before amnion was applied
amniotic membrane as a physiologic wound dressing. Arch Surg 1974.
109: 370-73.
biopsy specimens contained very little factor VIII in the
11. Matthews DN. Storage of skin for autogenous grafts. Lancet 1945; i:
vascular endothelial cells (fig. 3A), and cells reacting
775-78. with this antiserum were diffusely distributed, often giv-
12. Bisgaard H. Ulcers and eczema of the leg. Copenhagen: Munksgaard, 1948.
13. Burger K. Kunstliche scheidenbildung mittels eihauten. Zentralblatt
ing a dull fluorescence in the vessel wall and surround-
Gynakol 1937; 2437-40. ing connective tissues. -However, all biopsy specimens
repair of conjunctival defects with fetal membranes. Arch
14. de Rotth A. Plastic taken after 5 days of amnion application contained bril-
Ophthalmol 1940; 23: 522-25. liant immunofluorescent granules within the endothe-
15. Matthews RN, Bennett JP, Faulk WP. A review of the role of amniotic mem-
brane in surgical practice. Submitted for publication, 1980. lium, and many more vessels were identified (fig. 3B).
 1157

Fig. 3-Immunofluorescence with rabbit anti-factor VIII anti-

Fig. I-Representative fields in H and E stained sections. body.
(A) Absence of factor VIII and hence of blood-vessels in pre-amnion
Paucity of vessels in a generally densely collagenised stroma in pre- biopsy, in contrast with (B) which shows many sharply defined, thin-
amnion biopsy specimen (A) as compared with numerous widely walled, patent, small blood-vessels in post-amnion specimen.
patent vessels in a much less dense stroma in post-amnion biopsy speci-
men (B).

consists of the predominantly single-celled layer of

Autografting.-These results are reported in an amnion epithelium and its underlying basement mem-
brane. This part of the sac is bathed by amniotic fluid
accompanying paper. 12
To obtain a controlled assessment of the effectiveness without actually being in contact with maternal tissue.19
of the use of human amnion as an adjunct in wound In our hands, the most striking effect of amnion on
the healing of chronic leg ulcers was the development of
healing, four patients were managed by more traditional
new vessels as observed grossly, histopathologically, and
approaches to leg ulcer therapy before they had amnion
application and autografting.12 Neither the gross nor the immunohistologically by antiserum to factor VIII as a
microscopical appearance of their lesions after their in- marker of endothelium .20 Vessel growth-promoting fac-
itial treatment displayed the favourable features of tors are said to be produced by tumour cells21 and by in-

healthy granulation tissue seen after 5 days of amnion compatible donor lymphocytes in graft-versus-host reac-
application. tions.22 Since human extra-embryonic membranes share
structural and functional properties with certain tumour
Discussion cells,23,24 the effects we observed may be due to amnion
angiogenic factors acting on the capillary endothelium.
Mammalian embryos come to lie within a fluid-filled Could these factors be isolated for use as therapeutic
sac that arises from extra-embryonic tissues. The sac is agents in wound healing?
composed of two principal layers." The chorion (cyto- Other possible features that might promote the effec-
trophoblast) which forms the outer aspect of the sac is tiveness of amnion as a biological dressing are its
in contact with maternal cells without being rejected.l8 reported bacteriostatic properties,25 a feedback regula-
The inner aspect, that tissue used in our present studies, tion of procollagen synthesis by collagen products in the
explanted amnion,26 and an as yet undefined contribu-
tory function of the intermediary culture step we use.
The usefulness of amnion as a biological dressing for
burns" and chronic leg ulcers12 suggests that it may
have a broader application.
This work was supported in part by the Medical Research Council,
the South East Thames Regional Health Authority, British Petroleum,
and the East Grinstead Research Trust. We are grateful to the Obstet-
rics Departments of Pembury and Crawley Hospitals for providing the
amniotic membranes.

Requests for reprints should be addressed to W. P. F.

REFERENCES

1. Davis JS. Skin transplantation with a review of 550 cases at the Johns Hop-
kins Hospital. Johns Hopkins Hosp Rep 1910, 15: 307-95.
2. Stern W. The grafting of preserved amniotic membrane to burned and ulcer-
ated skin surfaces substituting skin grafts. JAMA 1913; 13: 973-74.
3. Burger K. Artificial vaginal reconstruction with the help of amnion. Zen-
Fig. 2-Reûculin-stained sections. tralblatt Fur Gynakol 1937, 2437-40.
4. Rötth A. de. Plastic repair of conjunctival defects with fetal membranes.
Pre-ammon biopsy specimens CA) demonstrate thick-walled vessels Arch Ophthalmol (Chicago) 1940; 23: 522-25.
1n a stroma containing coarse and often closely packed fibres; post- 5. Gruss JS, Jirsch DW. Human amniotic membrane: a versatile wound dress-
amnion biopsy (B) shows thinner walled, more sharply defined vessels ing. Can Med Assoc J 1978; 118: 237-40.
in a much looser interstitium with fibres that are fine and more widely
sennt-HTf
1158

REVERSIBLE MILD DIABETES IN CHILDREN

AFTER TREATMENT WITH
CHLORPROPAMIDE

WILLIAM J. MUTCH JOHN M. STOWERS

Diabetic Department, Aberdeen Royal Infirmary, Woolmanhill,
Aberdeen AB9 1GS

Summary 9 patients with juvenile-onset

In
chemical diabetes treated with oral
chlorpropamide, oral or intravenous assessments of car-
bohydrate tolerance were made regularly three weeks
after withdrawal of therapy. 6 patients with sequential
intravenous tests achieved statistically significant rever-
sal of their carbohydrate intolerance and have remained
normal for an average of 5.6 years (range 1-11 years). 2
patients who subsequently required insulin therapy were
maintained in remission for 3.5 years and 5 years,
respectively. There appears to be a group of young
patients with chemical diabetes who achieve significant
remission with sulphonylurea therapy. All 9 patients were given dietary advice and started on chlor-
propamide 50-100 mg daily. At presentation the weight of 6
Introduction patients was less than 1 standard deviation above the 50th per-
SULPHONYLUREAS "cure" experimental diabetes in centile, that of 2 less than 2 SD, and that of 1 more than 2 SD
above the 50th percentile.8 Glucose tolerance was tested yearly
animals.1 Fajans and Conn2 have shown improvement in
the glucose-tolerance tests of young patients with mild
after discontinuation of the chlorpropamide
for three weeks in
order to obviate any direct pharmacological effect. The dosage
diabetes after a course of tolbutamide. Various reports
of chlorpropamide was adjusted according to the result. The
have confirmed this.3,4 StowersS observed that patients
mean dosage during the period of follow-up was 158 mg daily
aged under 35 with chemical diabetes had a better (range 50-375 mg). The mean follow-up period was 7.2 years
carbohydrate-tolerance response to long-term chlor- (range 3-16 years). 4 patients had first-degree relatives with
propamide than did older patients. The former group diabetes; all the affected relatives were treated with tablets and
included a number of children aged 3 to 16 years.
We now describe the behaviour of carbohydrate 1 possibly had maturity-onset diabetes of the young (MODY).’
tolerance in a group of patients with juvenile-onset
chemical diabetes treated with oral chlorpropamide. Results
The behaviour of each patient’s carbohydrate
Patients and Methods tolerance is shown in table u. 7 patients achieved reversal
to normal tolerance. Of these, 6 have remained normal
9 patients (5 girls and 4 boys) with an average age at presen- for an average of 5.6 years (range 1-11 years), 3 of them
tation of 12.0 years (range 3-16) were reviewed (table I). Each having been off chlorpropamide for 8.0 years on average.
patient had an oral or intravenous glucose-tolerance test under In the other patient diabetes developed after a period of 7
basal conditions. The result of the intravenous test was years with increasing weight and intermittently raised
expressed numerically as an increment index.6 This is a fasting plasma-glucose levels. He defaulted and has
measure of the rate of fall of the increment of the plasma recently returned to the clinic, weighing 118 kg. Oral
glucose over the mean fasting level. An increment index >2.97 glucose tolerance is now normal 4 years after the finding
was normal and >2.46 abnormal. Results between these levels of overt diabetes. Throughout he has been free of
were regarded as probably abnormal. All patients had chemical diabetic symptoms and he is the only patient in this
diabetes at the time of diagnosis.7 group to have exceeded 2 SD above the 50th percentile

6. Eldad A, Stark N, Anais D, Golan T. Ben-Hur N. Amniotic membranes as Allergy 1972; 16: 9-39.
a biological dressing. SA Mediese Tydskrif 1977; 51: 272-75. 17. Boyd JD, Hamilton WJ. The human placenta. Cambridge: W. Heffer & Sons
7. Notea E, Hirschowitz B, Karev A, Mahler D. The use of lyophilised amnion Ltd. 1970: 22.
to treat burns and skin defects. Harefuah 1975; 88: 265-67. 18. Bourne G. The human amnion and chorion. London: Lloyd-Luke (Medical
8. Robson MC, Krizek TJ. Amniotic membranes as a temporary wound dress- Books) Ltd. 1962; 10.
ing. Surg Gynecol Obstet 1973; 136: 904-906. 19. Hoyes AD. Structure and function of the amnion. Ann Obstet Gynæcol 1975;
9. Dino BR, Eufemio GG, De Villa MS. The establishment of an amnion bank 4: 1-38.
and its practical application in surgery. J Phillipine Med Assoc 1966; 42: 20. Folkman J, Haudenschild CC, Zetter BR. Long-term culture of capillary
357-66. endothelial cells. Proc Nat Acad Sci U.S.A. 1979; 76: 5217-21.
10. Kirschbaum SM, Hernandez JH, Castellanas U. Use of amnion in extensive 21. Phillips P, Steward JK, Kumar S. Tumour angiogenesis factor in human and
burns. In: Broadbent TR. Proceedings of the IIIrd international congress animal tumour. Int J Cancer 1976; 17: 549-58.
in plastic surgery. Amsterdam: Excerpta Medica, 1963: 152-62. 22. Auerbach R, Sidky YA. Nature of the stimulus leading to lymphocyte in-
11. Bose B. Burn wound dressing with human amniotic membrane. Ann Roy duced angiogenesis. J Immunol 1979; 123: 751-54.
Coll Surg Eng 1979; 61: 444-47. 23. Faulk WP, Galbraith GMP. Trophoblast transferrin and transferrin recep-
12. Bennett JP, Matthews RN, Faulk WP. The treatment of chronic ulceration tors in the host-parasite relationship of human pregnancy. Proc Roy Soc
of the legs with human amnion. Lancet 1980; i: 1153. (B) 1979; 204: 83-97.
13. Burgos H, Faulk WP. The maintenance of amniotic membranes in culture. 24. Faulk WP, Yeager C, McIntyre JA, Ueda M. Oncofetal antigens of human
14. Gordon H, Sweet HH. A simple method for the silver impregnation of reticu- trophoblast. Proc Roy Soc (B) 1979; 206: 163-82.
lin. Am J Pathol 1936; 12: 545-51. 25. Robson MC, Krizek TJ. The effect of human amniotic membranes on the
15. Faulk WP, Johnson PM. Immunological studies of human placentæ: Identifi- bacterial population of infected rat burns. Ann Surg 1973; 177: 144-49.
cation and distribution of proteins in mature chorionic villi. Clin Exp Im- 26. Murphy WH, van der Mark K, McEneany LSG, Bornstein P. Charactenza-
munol 1977; 27: 365-75. tion of procollagen derived peptides unique to the precursor molecule. Bio-
16. Faulk WP, Hijmans W. Recent developments in immunofluorescence. Progr chemistry, NY 1975; 14: 3243-50.