Wound dressing

A method of treating a wound or burn which comprises covering the surface of the wound or burn with a cross-linked amnion dressing.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention pertains to methods and materials for treating wounds 
whether accidentally caused (trauma, burns, etc.) or intentionally created 
by surgery. In particular, it pertains to the preparation of amnion 
membranes and methods for treating burns and wounds with this material. 
2. Description of the Prior Art 
Any injury or surgical procedure that leaves a portion of the body devoid 
of skin places the organism in dire jeopardy with respect to survival. 
Essential body fluids escape through large open wounds. Similarly, 
pathological organisms (fungi, bacteria, microplasms, etc.) easily enter 
the body through these same sites. 
In most cases, wounds are closed by either: physically bringing the margins 
together vis-a-vis suture material (healing by primary intention) or by 
allowing the trauma site to heal naturally with new skin growing out and 
over the wound surface (healing by secondary intention). If the body is 
able to quickly close the wound opening before pathogens can invade and/or 
otherwise enter the tissues then the probability of survival is high. 
However, if wound closure is slow, then the chances of sepsis is high. 
If a wound is so large as to prevent the approximation of its margins using 
sutures, then a dressing has to be used to temporarily seal off the wound 
from the surrounding environment. The ideal wound dressing should be thin, 
flexible and inert; provide adequate gas exchange; adhere tightly to the 
wound surface; prevent microbial invasion; surpress fluid loss; offer a 
template for new skin growth; should be readily available, easily stored, 
easily used, relatively cheap, and widely distributed. 
The problem of providing a suitable wound dressing has existed since the 
time when the first open wounds were dressed or covered in order to 
facilitate healing. The intellectual search for the ideal wound dressing 
has probably been underway for the past 250 years. Its significance has 
been recognized for a similarly long period of time because nearly all 
large (greater than 40% total surface area) burn injuries led to death by 
bacterial sepsis prior to 1943. The chances for survival were largely 
improved when the hypochlorite bathing solution was first used 
(Bunyan-England 1940/1943) the advent of antibiotics and later when the 
pig skin temporary wound dressing was introduced. 
Currently, Open wounds are: 
(1) left uncovered and/or untreated; 
(2) covered with an anti-bacterial ointment (i.e. silversulphadiazine); 
(3) temporarily covered with pigskin; 
(4) temporarily covered with fresh amnion; 
(5) covered by homographic graft from other areas of the body; 
(6) covered with heterographic skin (from mother, father, brother, sister, 
etc.) 
(7) covered with a cloth or non-biological membrane or bandage; or 
(8) any combination of the above. 
None of these are totally acceptable. Pigskin sloughs off after twenty to 
twenty-five days due to an antigen/antibody reaction. Fresh amnion can be 
enzymatically digested by the host in three to four days if the area 
covered is moist. Amnion may also evoke an antigen-antibody reaction and 
there is also a risk of transferring pathogens from donor to recipient. 
Homographic skin grafts cover one area while opening up another at the 
donor site. Heterographic skin is usually rejected by the patient in six 
to twenth-five days (another antigen/antibody reaction). 
Because human amnion, being a continuation of the fetal integument, has 
many of the natural and physical qualities of skin and is relatively cost 
effective, it has become more popular as a temporary dressing than porcine 
xenografts in some burn treatment institutions. The successful use of 
fresh and/or frozen amnion as a biological dressing (i.e. on burn wounds), 
depends in part on its structure an integrity as a microbial barrier and 
on the sterility of the membrane, as well as the condition and nature of 
the wound to which it is applied. 
Although amnion is a popular temporary dressing, in many ways it is even 
more temporary than pigskin or heterographic skin. Electronmicroscopy 
reveals the near total degradation of amnion by the host as early as three 
days post application if the covered site remains moist. The degradation 
appears to be due to digestion by enzymes in the wound exudate observable 
as early as twenty-four hours after application. Ultra-structural features 
of the amnion dermis, epidermis and basal lamina are completely lost after 
forty-eight hours on the patient. After seventy-two hours, the membrane 
components are difficult if not impossible to discern. Thus, the barrier 
qualities of the sterile fresh amnion may be lost long before the 
patient's need for barrier protection has ended. It is now recommended 
that, when used on severe wounds, the amnion be replaced at approximately 
three day intervals. In addition, electron microscopy discloses that some 
of the collagen filaments in the amnion dermis are partially unraveled 
during sterilization procedures (which usually empoloys a sodium 
hypochlorite solution) required to render the amnion asceptic which 
further compromises the barrier quality of the membrane even prior to its 
application to the wound. 
SUMMARY OF THE INVENTION 
In the present invention, a wound dressing is provided from amnion, treated 
in such a way that it becomes a substantially more permanent dressing 
until the wound is healed. Fresh amnion from any animal source is fixed or 
stabilized by glutaraldehyde solutions or any other fixing or tanning 
solution so that the proteins thereof are cross-linked. Due to such change 
in the physical nature of the proteins comprising the structure of the 
amnion, the host's enzymes can no longer digest or break it down. Its 
physical properties (strength, flexibility, etc.) are also improved. Other 
objects and advantages of the invention will be understood from reading 
the specification which follows.

DESCRIPTION OF THE INVENTION 
To prepare a wound dressing according to the present invention, amnion is 
collected from any suitable animal species (human, cattle, pigs, etc.) 
from term or near term fetuses at the time of delivery or slaughter. The 
amnion is excised away from the umbilical cord, freed of chorion material 
by gentle scraping and rinsed with sterile saline. Alternately the chorion 
could be removed by gentle scraping after fixation to be described 
hereafter. All processing should be carried out under sterile or 
semisterile conditions. 
The amnion is then fixed in a suitable fixing solution for a period of time 
from a few hours to a few days, depending upon the type of fixing solution 
and the concentration used. Typical fixing agents can be selected from the 
group consisting of glyoxal, glutaraldehyde, hydroxyadipaldehyde, pyruvic 
aldehyde, crotonaldehyde, acetaldehyde, acrolein, methacrolein, 
formaldehyde, malonaldehyde, succinaldehyde, and chromic acid. 
Combinations of fixatives may be used. For instance, 0.6%, 3% or 6% 
glutaraldehyde solutions have been found to sufficiently fix the tissue in 
twenty-four to forty-eight hours. However, it appears that a range of from 
0.001% to 25% concentrations of glutaraldehyde are workable, the stronger 
solutions requiring a fixation time of from two or three hours and the 
weaker solutions requiring three or four weeks. Excessive fixation does 
not appear to affect the quality of the membrane. 
The minimum ratio of fixing solution volume to amnion volume should also be 
considered. In a 0.6% glutaraldehyde solution, a ratio of around fifty is 
required. Of course, the ratio is smaller for greater solution 
concentrations (25% glutaraldehyde a ratio of 1-5) and larger for smaller 
concentration solutions (0.001% a ratio of 1000). 
The fixing solution should be buffered with a buffer that produces a 
stabilized membrane that is compatible with life, i.e. phosphate, 
bicarbonate, barbital, etc. The buffer should be in the range of 0.001 to 
0.2 m (mols), and have a pH of 6.5 to 8.4. The osmolarity of the solution 
should ideally be around isotonic conditions (300 mOs) or perhaps even 
slightly hypertonic (600 mOs). However, a range of 50 to 2,000 mOs is 
acceptable depending upon solution concentrations. The osmolarity of the 
fixative solution is usually determined by the fixative and buffer 
concentrations. Inert materials (salts, sugars, etc.) may also be added to 
the solution in order to increase its osmolarity. 
After fixing, the amnion is prepared for storage. It may be stored in a 
liquid phase, air-dried or lyophilized (freeze dried). If stored in 
solution, the amnion may be shaped into utilizable pieces, rolled in Nylon 
mesh gauze and transferred to a maintenance solution of the same fixing 
materials, i.e. 0.6% to 3.0% glutaraldehyde solution with buffer or any 
other antiseptic solution, saline or even distilled water. The material is 
then placed in sterile containers and stored preferably at 0.degree. to 
4.degree. C., but up to 25.degree. C. 
Anatomically, the stabilized amnion material is found to exist in three 
parts, the epidermal layer, the basement membrane, and an underlying 
connective tissue layer similar to the dermis of skin. The epidermis is 
comprised of a single layer of cuboidal cells closely attached to one 
another. After fixation (chemical stabilization) the cells are no longer 
viable. The epidermis rests on a basement membrane layer or basal lamina 
which is comprised of a fibrillar proteinacious layer approximately three 
to five microns thick. This membrane is for the most part intact without 
obvious voids which would permit pathogen invasion. The underlying 
connective tissue (comprised of a compact layer, a fibroblast layer, and a 
spongy layer) but for simplicity's sake henceforth referred to as the 
amnion dermis is comprised of a layer of tightly packed collagen filaments 
fused into a solid mass of fibers. Under this solid fiber mat are to be 
found a looser arrangement of collagen fibers and filaments. All of the 
proteins in the amnion are cross-linked by the stabilization process. 
Occasionally fibrocytic cells can be found in this layer. Hence, the 
cells, the interstitial materials (collagen, ground substances, etc.) and 
the occasional vascular components (small capillary remnants in the deep 
dermis) are chemically fixed or precipitated by the stabilizing or fixing 
solution or process. 
The stabilized amnion material, after fixation, shows a increase in tensile 
strength while not being appreciably changed with respect to structural 
organization, thickness, coloration, etc. No dermal appendages (glands, 
hair follicles, etc.) or holes are normally found in the amnion. It is a 
homogeneous biological membrane. 
After preparation, the amnion prosthesis can be brought from the storage 
fluid and rinsed several times using sterile saline. Minimum rinsing 
changes, times and volumes will depend on the amount of amnion material 
used and the type of storage solution employed. However, the rinses are 
intended to reduce the free glutaraldehyde (or other fixative) 
concentration in the final solution to an acceptable human allowance. The 
rinses should be carried out under sterile conditions. 
If the stabilized amnion is to be air-dried, it would be rinsed after 
fixation in water and dehydrated through a series of alcohol ending in a 
100% ethyl alcohol bath or any other acceptable organic solvent such as 
freon, acetone, etc. Then the amnion membrane would be air-dried, packaged 
and sterilized. It may be sterilized in an appropriate gas, i.e. ethylene 
dioxide or formaldehyde gas, or with high levels or gamma radiation (i.e. 
2,000,000 rads). 
The fixed amnion could also be dried by lyophilization procedures which are 
ideal for biological wound dressings. In such procedures, the fixed amnion 
would be rinsed with saline and then water followed by cryogenic freezing, 
i.e. with liquid nitrogen at -185.degree. C. The frozen material is then 
subjected to high vacuum causing the water to sublimate. Sterilization 
would be the same as in air-drying. 
To use on the wound, the hydrated amnion prosthesis is unrolled, removed 
from the Nylon mesh netting, if there is such, placed over the wound 
surface (the amnion dermis placed downward) and held in place by 
bandaging, sutures or allowed to attach by wound serum dessication 
(coagulation). When dried amnion is used, it quickly imbibes the plasma 
from the wound surface, evokes platelet activity and becomes tightly 
attached. 
The prosthesis can be used on any open wound that has been properly cleaned 
and debrided. The wounds can be full-thickness skin loss caused by: 
excisional trauma, abrasion, thermal or chemical trauma or surgical 
excision. Similarly, the material can be used on wounds that are intended 
to heal by means of secondary intention (wounds produced by pilonidal cyst 
excision, fistulectomies, etc.). How the amnion is first physically 
attached (i.e. adherence, sutures, etc.) to the wound is an unimportant 
aspect, but efforts should be made to allow the prosthesis to stabilize on 
the wound surface for the first few hours to allow its complete 
attachment. In most cases, the amnion quickly and tenaciously attaches to 
the wound surface; especially when dried or lyophilized membrane is used. 
Underlying wounds are then allowed to heal under the amnion covering. 
Surveillance should be maintained for any obvious pockets of purulent 
material that may form under the amnion although this is rare. As the 
membrane is extremely thin and nearly transparent, surveillance is easy. 
Should localized areas of contamination form, then the amnion can easily 
be excised away from these small areas, the wound cleansed in that area 
and a smaller stabilized amnion patch used. 
The amnion appears to detach from the wound after epithelization and 
keratinization has taken place. In very large surface wounds, mesh 
homographic skin tissue should be situated between the wound surface and 
the amnion covering to provide a source for epidermal cells and to 
accelerate the process of total epithelization. 
The fixed amnion dressing can be used for long periods without need of 
replacement or fear of eliciting a host/graft response. This is due to the 
resultant inertness of the material after fixation. The dermis of the 
stabilized amnion also provides a pattern for the synthesis, deposition 
and orientation of the host's new dermis (collagen fibers). The amnion 
provides a suitable wound barrier which cannot be compromised by enzymatic 
degradation either mediated by the host or by invasive microorganisms. The 
amnion membrane is devoid of hair follicles, glands, etc. whose structural 
configuration would otherwise provide crypts, folds and/or passages 
through which microbial access would be provided to the wound environment. 
It provides a suitable mechanical and water barrier to the wound tissue 
and as such provides structural integrity and prevents excessive fluid 
loss. The stabilized amnion skin prosthesis (in that it is fetal skin) is 
the perfect wound dressing and/or temporary skin. Additionally, fixation 
reduces the risk of pathogen transfer from donor to recipient to an 
extremely low level or even zero. Viruses, bacteria and fungi that 
normally may reside in an amnion are all killed by the processing 
procedure. 
The fixed amnion prosthesis used as a wound dressing is a substantial 
advancement in the wound healing art in that the material remains in 
place, intact and uncompromisable until the recipient host tissue (wound) 
no longer needs it and it simply falls off. It does not and cannot evoke a 
host/graft response (antigen/antibody reaction) and therefore does not 
seem to influence the recipient's immune system. It appears to be 
biologically inert and as such is safer than synthetic membranes that 
might contain unsafe organic compounds. The physical nature of the 
stabilized amnion material encourages would repair by offering a template 
upon which new collagen fibers can be laid down. The strength of the 
stabilized amnion material also contributes to the integrity of underlying 
delicate wound healing tissues and as such appears to suppress wound 
contraction. 
The epidermal cells of the host tissue grow between the stabilized amnion 
material and the wound surface. Upon differentiation of the newly formed 
epidermal layer and the keratinization of this layer of cells, the 
stabilized amnion material is dessicated and as such is quietly released 
and falls off. No invasive procedures are required after the stabilized 
amnion material is in place. Pain in the wound is immediately attenuated 
and/or alleviated after the membrane is applied. 
Unexpected properties suggest that stabilized amnion material may be used 
in other areas of biological endeavor. Items such as artificial tempanic 
membranes, tendons, ligaments, etc. may be produced from this inert 
material. Unexpected results include leukocytic aggregation at the surface 
of the stabilized amnion material after placement on the wound. These 
cells appear to be involved in a benign foreign body reaction. A layer of 
leukocytes provides an additional barrier to pathogen invasion, as well as 
generating an adherence of viable cells to the inert stabilized amnion 
material. This reaction also appears to keep the material hydrated through 
the course of repair with dehiscence occurring in the stabilized amnion 
material in those areas after the wound has healed (epidermis forms, 
differentiates, and keratinizes). 
Experiments suggest that the stabilized amnion material is antigenically 
inert. When placed subcutaneously in a recipient, it evokes only a foreign 
body reaction (becomes encapsulated) but does not produce a typical 
immulogical response. Ultrastructurally, the stabilized amnion remains 
intact when placed on the surface of experimental wounds, burns and in the 
subcutaneous capsules for long periods of time. Healing tissue under the 
amnion cover appears to be at least equal to but more commonly better than 
that observed in untreated wounds. For instance the dermis is thicker, 
more ordered and observed to be structurally more normal and the size of 
the wound is not diminished by wound contraction. 
Millions of individuals are burned and severely wounded annually throughout 
the world. Burns and wounds requiring a wound dressing are all subjects 
for the stabilized amnio material prosthesis of the present invention. The 
stabilized amnion material prosthesis would stay in place until 
reepithelization, grafting, etc. had taken place, not being compromised by 
the host or microbes and maintaining its integrity until the wound has 
healed. The stabilized amnion material is tough, flexible and durable. 
The fresh human amnion and pigskin currently used as temporary wound 
dressings are expensive to obtain and prepare, difficult to store and are 
of limited availability. In comparison, the stabilized amnion material can 
be produced easily and cheaply, stored in small packages (in dry or 
hyrated states) under normal refrigeration (materials stored in fixative 
solutions) or even unrefrigerated for ready distribution and employment 
not only in burn wards, hospitals and medical centers, but by clinicians 
anywhere at any time. In addition to being used as a wound dressing, it 
can be used for artificial fascia, artificial tendons, supportive 
ligaments, tempanic membranes or anywhere an artificial membrane might 
provide a support, partition, isolation or structure for the recipient. 
While a few variations of the invention have been described herein, many 
others may be made without departing from the spirit of the invention. 
Accordingly, it is intended that the scope of the invention be limited 
only by the claims which follow.