Chemical treatment, without detergents or enzymes, of tissue to form an acellular, collagenous matrix

The invention is directed to collagenous tissues which have been treated to remove non-collagenous components such as cells, cellular debris, and other extracellular matrix components, such as proteoglycans and glycosaminoglycans, normally found in native tissues. Treatment of the tissue with alkali, chelating agents, acids and salts removes non-collagenous components from the collagenous tissue matrix while controlling the amount of swelling and dissolution so that the resultant collagen matrix retains its structural organization, integrity and bioremodelable properties. The process circumvents the need to use detergents and enzymes which detrimentally affect the cell compatibility, strength and bioremodelability of the collagen matrix. The collagenous tissue matrix is used for implantation, repair, or use in a mammalian host.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention is in the field of tissue engineering. The invention is 
directed to collagenous tissues which have been treated to remove 
non-collagenous components such as cells, cellular debris, and other 
extracellular matrix components, such as proteoglycans and 
glycosaminoglycans, normally found in native tissues. Treatment of the 
tissue with alkali, chelating agents, acids and salts removes 
non-collagenous components from the collagenous tissue matrix while 
controlling the amount of swelling and dissolution so that the resultant 
collagen matrix retains its structural organization, integrity and 
bioremodelable properties. The process circumvents the need to use 
detergents and enzymes which detrimentally affect the cell compatibility, 
strength and bioremodelability of the collagen matrix. The collagenous 
tissue matrix is used for implantation, repair, or use in a mammalian 
host. 
2. Brief Description of the Background of the Invention 
The field of tissue engineering combines the methods of the engineering 
with the principles of life sciences to understand the structural and 
functional relationships in normal and pathological mammalian tissues. The 
goal of tissue engineering is the development and ultimate application of 
biological substitutes to restore, maintain or improve tissue functions. 
[Skalak, R. and Fox, C. F., "Tissue Engineering", Alan R. Liss Inc. N.Y. 
(1988)] 
Collagen is the principal structural protein in the body and constitutes 
approximately one-third of the total body protein. It comprises most of 
the organic matter of the skin, tendons, bones and teeth and occurs as 
fibrous inclusions in most other body structures. Some of the properties 
of collagen are its high tensile strength; its ion exchanging ability, due 
in part to the binding of electrolytes, metabolites and drugs; its low 
antigenicity, due to masking of potential antigenic determinants by the 
helical structure, and its low extensibility, semipermeability, and 
solubility. Furthermore collagen is a natural substance for cell adhesion. 
These properties and others make collagen a suitable material for tissue 
engineering and manufacture of implantable biological substitutes and 
bioremodelable prostheses. 
As collagen is one major component of these biological substitutes, a 
method for obtaining sufficient quantities of collagen that is consistent 
in quality is needed. A need currently exists for an improved method for 
the removal of non-collagenous components such as cells, cellular debris, 
and other extracellular matrix components, such as proteoglycans and 
glycosaminoglycans, normally found in native tissues to yield a 
substantially pure native collagen matrix. Some of these non-collagenous 
structures that are present in native tissues are believed to be antigenic 
and will elicit a chronic inflammatory response when implanted in a host. 
However, in the art there are a variety of methods for the cleaning of 
such collagenous tissue which have resulted in collagenous compositions 
with different characteristics. The method used should be one that 
maintains the biological and physical properties of collagen and 
collagenous tissues suitable for use in tissue engineering. 
In the art of treating a collagenous tissue to yield essentially a 
collagenous matrix, detergents and surfactants have customarily been used 
in the extraction of cells and lipids from the tissue. Detergents such as 
sodium dodecyl sulfate (SDS) are amphipathic molecules wherein the 
hydrophobic region binds to protein and are believed to increase the 
negative charge of the protein. When implanted, the increase in charge 
results in both the swelling of the tissue due to increased water binding 
by the hydrophilic region of the molecule, and decreased thermal stability 
in collagen by disrupting hydrogen bonding. Swelling both opens the 
structure of the collagen molecule making it susceptible to cellular 
enzymes such as collagenase and destabilizes the collagen matrix to result 
in a weakened construct. (Courtman, et al. Journal of Biomedical Materials 
Research 1994; 28:655-666.) It is further believed that SDS residues 
remain bound to the collagen and prevent cells from migrating into the 
implant. (Wilson, G J et al. Ann Thorac Surg 1995; 60:S353-8. Bodnar E, et 
al. "Damage of aortic valve tissue caused by the surfactant sodium dodecyl 
sulfate." Thorac Cardiovasc Surg 1986; 34:82-85.) Because detergents used 
in a chemical cleaning method can undesirably bind to and alter the 
bioremodeling capabilities of collagen in the treated tissue, the 
inventors have developed a method that eliminates the need for detergents. 
Chemical cleaning of tissue with enzymes such as trypsin, pepsin and 
collagenase is known in the art but their use will result in chemical 
modification of the native collagen molecules and will adversely affect 
the structural integrity of the construct. Enzyme treatment of collagenous 
tissue is known in the art for removal and/or modification of 
extracellular matrix associated proteins. Proteases such as pepsin, 
trypsin, dispase, or thermolysin are used in the removal of collagen 
telopeptides to yield atelopeptide collagen. Collagen telopeptides are the 
non-triple helical portion of the collagen molecule and have been thought 
by some researchers to be weakly antigenic while by others they are 
thought to be responsible for the strong mechanical properties of 
collagen. Limited digestion of collagenous tissue will remove telopeptides 
without dissociation of the collagen matrix of the tissue, while prolonged 
digestion will dissociate the collagen fibrils into atelopeptide collagen 
monomers. It is also known in the art to modify and remove nucleic acids 
from the matrix using enzymes that digest endogenous RNA and DNA through 
use of RNAse and DNAse, respectively. As treatment with enzymes can affect 
the structural integrity of the collagen, the present method of the 
invention circumvents their use. 
Methods for obtaining collagenous tissue and tissue structures from 
explanted mammalian tissue, and processes for constructing prostheses from 
the tissue, have been widely investigated for surgical repair or for 
tissue and organ replacement. The tissue is typically treated to remove 
potentially cytotoxic cellular and noncollagenous components to leave a 
natural tissue matrix. Further processing, such as crosslinking, 
disinfecting or forming into shapes have also been investigated. Previous 
methods for treating collagenous tissue to remove tissue components from 
the organized tissue matrix have employed detergents, enzymes or promote 
uncontrolled swelling of the matrix. WO 95/28183 to Jaffe, et al. 
discloses methods to decrease or prevent bioprosthetic heart valve 
mineralization postimplantation. The disclosed methods provide biological 
material made acellular by controlled autolysis. Autolysis is controllably 
performed using at least one buffer solution at a preselected pH to allow 
autolytic enzymes present in the tissue to degrade cellular structural 
components. U.S. Pat. No. 5,007,934 to Stone and, similarly, U.S. Pat. No. 
5,263,984 to Li, et al. both disclose a multiple step method for chemical 
cleaning of ligamentous tissue. The method utilizes a detergent to remove 
lipids associated with cell membranes or collagenous tissue. U.S. Pat. No. 
5,523,291 to Janzen, et al. discloses an comminuted injectable implant 
composition for soft tissue augmentation derived from ligamentum nuchae. 
The ligament is treated with a series soaks in a strongly alkaline 
solution of sodium hydroxide followed by hydrochloric acid solution and 
then sodium bicarbonate. U.S. Pat. No. 5,028,695 to Eckmayer, et al. 
discloses a process for the manufacture of collagen membranes in which 
collagenous tissue is repeatedly treated with a strong alkali and 
subsequently with a strong acid for a number of times then further treated 
with inorganic saline treatment to shrink the membranes and then with 
solvent to dry them. 
SUMMARY OF THE INVENTION 
Bioremodelable collagenous tissue matrices and methods for chemical 
cleaning of native tissue to produce such tissue matrices are disclosed. 
The present invention overcomes the difficulties in obtaining 
bioremodelable tissue matrices that are substantially collagen. The 
invention provides tissue matrices that can be used as a prosthetic device 
or material for use in the repair, augmentation, or replacement of damaged 
and diseased tissues and organs. 
The chemical cleaning method of this invention renders biological material, 
such as native tissues and tissue structures, substantially acellular and 
substantially free of non-collagenous components while maintaining the 
structural integrity of the collagenous tissue matrix. As detergents are 
not used in the chemical cleaning process, detergent residues that would 
normally remain bound to the tissue matrix are not present. As enzymes are 
not used, the collagen telopeptides are retained on the collagen 
molecules. The method comprises contacting a normally cellular native 
tissue with a chelating agent at a basic pH, contacting the tissue with 
salt solution at an acidic pH, contacting the tissue with a salt solution 
at a physiologic pH, and, then finally rinsing the resultant chemically 
cleaned tissue matrix. 
This invention is directed to a chemically cleaned tissue matrix derived 
from native, normally cellular tissues. The cleaned tissue matrix is 
essentially collagen with intact rendered substantially free of 
glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous 
proteins and nucleic acids such as DNA and RNA. Importantly, the 
bioremodelability of the tissue matrix is preserved as it is free of bound 
detergent residues that would adversely affect the bioremodelability of 
the collagen. Further the collagen is telopeptide collagen as the 
telopeptide regions of the collagen molecules remain intact as it has not 
undergone treatment or modification with enzymes during the cleaning 
process. 
The collagenous material generally maintains the overall shape of the 
tissue it is derived from but it may be layered and bonded together to 
form multilayer sheets, tubes, or complex shaped prostheses. The bonded 
collagen layers of the invention are structurally stable, pliable, 
semi-permeable, and suturable. When the matrix material is implanted into 
a mammalian host, undergoes biodegradation accompanied by adequate living 
cell replacement, or neo-tissue formation, such that the original 
implanted material is ultimately remodeled and replaced by host derived 
tissue and cells. 
It is, therefore, an object of this invention to provide a method for 
cleaning native tissue resulting in a tissue matrix that does not exhibit 
many of the shortcomings associated with many of the methods developed 
previously. The method effectively removes non-collagenous components of 
native tissue without the use of detergents or enzymes to yield a tissue 
matrix comprised substantially of collagen. 
Another object is the provision of a bioremodelable tissue matrix material 
that will allow for and facilitate tissue ingrowth and/or organ 
regeneration at the site of implantation. Prostheses prepared from this 
material, when engrafted to a recipient host or patient, concomitantly 
undergoes controlled bioremodeling and adequate living cell replacement 
such that the original implanted prosthesis is remodeled by the patient's 
living cells to form a regenerated organ or tissue. 
Still another object of this invention is to provide a method for use of a 
novel multi-purpose bioremodelable matrix material in autografting, 
allografting, and heterografting indications. 
Still a further object is to provide a novel tissue matrix material that 
can be implanted using conventional surgical techniques.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a method for processing native collagenous 
tissues for transplantation. The processing method is designed to generate 
an implantable, graftable collagenous biological tissue material, an 
extracellular matrix comprising collagen, that serves as a scaffold that 
can be bioremodeled by a host in vivo or by living cells in culture in 
vitro. 
This invention is further directed to a tissue engineered prostheses formed 
from processed native collagenous tissue, which, when implanted into a 
mammalian host, can serve as a functioning repair, augmentation, or 
replacement body part, or tissue structure, and will undergo controlled 
biodegradation occurring concomitantly with remodeling by the host's 
cells. The tissue matrix can be used as a prosthetic material for 
autografting, allografting, and heterografting indications. The prosthesis 
of this invention, in its various embodiments, thus has dual properties: 
First, it functions as a substitute body part, and second, while still 
functioning as a substitute body part, it functions as a remodeling 
template for the ingrowth of host cells. Although the prostheses will be 
illustrated through construction of various devices and constructs, the 
invention is not so limited. It will be appreciated that the device design 
in its material, shape and thickness is to be selected depending on the 
ultimate indication for the construct. 
The chemical cleaning method of this invention renders biological material, 
such as native tissues and tissue structures, substantially acellular and 
substantially free of non-collagenous components while maintaining the 
structural integrity of the collagenous tissue matrix. Elastin is 
sometimes present in native tissue in small amounts and is not removed by 
the chemical cleaning method. The presence of elastin may be desirable for 
certain applications. As used herein, the term, "substantially acellular" 
means having at least 95% fewer native cells and cell structures than the 
natural state of the biological material. "Cells and cellular structures" 
refer to cells, living or not living, cell remnants, cell membranes and 
membrane structures. By use of the term, "substantially free of 
non-collagenous components", means that glycoproteins, glycosaminoglycans, 
proteoglycans, lipids, non-collagenous proteins and nucleic acids such as 
DNA and RNA comprise less than 5% of the resultant tissue matrix. As 
detergents are not used in the chemical cleaning process, detergent 
residues that would normally remain bound to the tissue matrix are not 
present. As enzymes are not used, the collagen telopeptides are retained 
on the collagen molecules. Further, the chemical cleaning method renders 
the biological material both sterile and endotoxin free when processed 
using sterile equipment, solutions and aseptic technique. 
The term, "structural integrity", refers to the capacity of the chemically 
cleaned collagenous tissue matrix to withstand forces such as tension, 
compression, and support The structural integrity of the biological 
material is preserved as swelling is minimized in the chemical treatment 
steps even though some swelling will occur during treatment. Uncontrolled 
or excessive swelling both opens the structure of the collagen molecule 
making it susceptible to cellular enzymes such as collagenase and 
destabilizes the collagen to result in a weakened construct. As swelling 
affects the intramolecular structure of the collagen molecule, it affects 
the overall structure of the material on a intermolecular level by 
disrupting the native crosslinks between collagen molecules. Together, the 
structure of the collagen molecule and the crosslinks between collagen 
molecules lend structural integrity to the material. 
Tissue matrix material that maintains much of its native structural 
integrity is useful, for instance, when used as a prosthetic device or as 
material to construct mulitilayered or complex devices. The integrity of 
the material is important if it is to perform a load bearing function such 
as a body wall support, a vascular device, or an orthopedic device. 
Related to structural integrity is the term "suturable" which means that 
the mechanical properties of the material includes suture retention which 
permits needles and suture materials to pass through the prosthesis 
material at the time of suturing of the prosthesis to sections of native 
tissue, a process known as anastomosis. During suturing, such prostheses 
must not tear as a result of the tensile forces applied to them by the 
suture, nor should they tear when the suture is knotted. Suturability of 
the prosthetic material, i.e., the ability of prostheses to resist tearing 
while being sutured, is related to the intrinsic mechanical strength of 
the prosthesis material, the thickness of the graft, the tension applied 
to the suture, and the rate at which the knot is pulled closed. 
Biological material as defined in the invention includes but is not limited 
to harvested mammalian tissues, and structures thereof, derived from 
human, bovine, porcine, canine, ovine, caprine, and equine organisms. 
Tissue structures such as dermis, artery, vein, pericardium, heart valve, 
dura mater, ligament, intestine and fascia are all preferred tissue 
structures that are able to be cleaned by the methods of this invention to 
yield a tissue matrix that is substantially acellular and substantially 
free of non-collagenous components. 
A preferred source of mammalian tissue is the tunica submucosa from small 
intestine, most preferably from porcine small intestine. In native small 
intestine, the tunic submucosa is the connective tissue layer of the organ 
and comprises both lymphatic and blood vessel cells. Methods for obtaining 
tunica submucosa are disclosed in WO 96/31157 and is incorporated herein. 
To obtain porcine tunica submucosa, also termed "submucosa", the small 
intestine of a pig is harvested and mechanically stripped, preferably by 
use of a gut cleaning machine (Bitterling, Nottingham, UK). The gut 
cleaning machine forcibly removes the fat, muscle and mucosal layers from 
the tunica submucosa using a combination of mechanical action and washing 
with water. The mechanical action can be described as a series of rollers 
that compress and strip away the successive layers from the tunica 
submucosa when the intact intestine is ran between them. As the tunica 
submucosa of the small intestine is comparatively harder and stiffer than 
the surrounding tissue, the softer components from the submucosa are 
removed from the tunica submucosa. The result of the machine cleaning is 
such that the mesenteric tissues, the tunica serosa and the tunica 
muscularis from the ablumen of the tunica submucosa and as well as the 
layers of the tunica mucosa from the lumen of the tunica submucosa are 
removed from the tunica submucosa so that the tunica submucosa layer of 
the intestine solely remains. The chemically cleaned tissue matrix of the 
tunica submucosa is also termed "intestinal collagen layer" or "ICL". It 
is noted that in some animal sources, such as carnivores and omnivores, 
the small intestine includes a stratum compactum which is also removed by 
this mechanical cleaning step. 
Other methods of mechanically stripping layers of the small intestine are 
known in the art as described in U.S. Pat. No. 4,902,508 to Badylak, 
incorporated herein by reference. The method disclosed by this patent 
includes mild abrasion of the intestinal tissue to remove the abluminal 
layers, including the tunica serosa and the tunica muscularis, and the 
inner layers consisting of at least the luminal portion of the tunica 
mucosa. The layers that remain are the tunica submucosa with the attached 
basilar layer consisting of lamina muscularis mucosa and, if initially 
present in the harvested mammalian tissue, stratum compactum. Intestinal 
material obtained by either method can be implanted or first formed into 
body wall or vascular device by a number of methods including suturing, 
stapling. adhesive compositions, chemical bonding and thermal bonding. 
Terms pertaining to certain operating parameters are defined for the entire 
specification and the examples for amounts, times and temperatures that 
can be varied without departing from the spirit and scope of the 
invention. As used herein, an "effective amount" refers to the volume and 
concentration of composition required to obtain the effect desired. A 
preferred effective amount for the chemical cleaning of tissue is a ratio 
of 100:1 v/v of solution to tissue but volumes more or less can be 
determined by the skilled artisan when considering the shape, bulk, 
thickness, density, and cellularity of the tissue to be cleaned. The time 
required for the chemical steps to be effective can be appreciated by 
those of skill in the art when considering the cellularity, matrix 
density, and thickness of the material to be cleaned. Larger, thicker, or 
denser materials will take longer for the solutions to penetrate and 
equilibrate in tissue. The temperatures for the environment and the 
solutions used in the present invention is preferably at ambient room 
temperature, about 25.degree. C., but can be anywhere in the range of 
above the freezing temperatures of the solutions used to less than the 
denaturation temperature of the tissue material being treated. 
Temperatures between about 4.degree. C. to about 45.degree. C. are 
sufficient for the cleaning treatment to be effective. Agitation is meant 
to be mechanical shaking or mixing and is used to improve the penetration 
of the chemical compositions into the tissue and to reduce the time needed 
for chemical treatment to be effective. The term "buffered solution" 
refers to an aqueous solution containing at least one agent which 
preserves the hydrogen ion concentration or pH of the solution. 
In the preferred method, harvested tissue may need to be cleaned manually, 
as by gross dissection, and/or mechanically cleaned of excess tissues such 
as fat and vasculature. Manual cleaning may be necessary for some tissues 
for handling manageability during processing or for most effective 
chemical treatment. 
The tissue is first treated by contacting the tissue with an effective 
amount of chelating agent, preferably physiologically alkaline to 
controllably limit swelling of the tissue matrix. Chelating agents enhance 
removal of cells, cell debris and basement membrane structures from the 
matrix by reducing divalent cation concentration. Alkaline treatment 
dissociates glycoproteins and glycosaminoglycans from the collagenous 
tissue and saponifies lipids. Chelating agents known in the art which may 
be used include, but are not limited to, ethylenediaminetetraacetic acid 
(EDTA) and ethylenebis(oxyethylenitrilo)tetraacetic acid (EGTA). EDTA is a 
preferred chelating agent and may be made more alkaline by the addition of 
sodium hydroxide (NaOH), calcium hydroxide Ca(OH).sub.2, sodium carbonate 
or sodium peroxide. EDTA or EGTA concentration is preferably between about 
1 to about 200 mM; more preferably between about 50 to about 150 mM; most 
preferably around about 100 mM. NaOH concentration is preferably between 
about 0.001 to about 1 M; more preferably between about 0.001 to about 
0.10 M; most preferably about 0.01 M. Other alkaline or basic agents can 
be determined by one of skill in the art to bring the pH of the chelating 
solution within the effective basic pH range. The final pH of the basic 
chelating solution should be preferably between about 8 and about 12, but 
more preferably between about 11.1 to about 11.8. In the most preferred 
embodiment, the tissue is contacted with a solution of 100 mM EDTA/10 mM 
NaOH in water. The tissue is contacted preferably by immersion in the 
alkaline chelating agent while more effective treatment is obtained by 
agitation of the tissue and the solution together for a time for the 
treatment step to be effective. 
The tissue is then contacted with an effective amount of acidic solution, 
preferably containing a salt. Acid treatment also plays a role in the 
removal of glycoproteins and glycosaminoglycans as well as in the removal 
of non-collagenous proteins and nucleic acids such as DNA and RNA. Salt 
treatment controls swelling of the collagenous tissue matrix during acid 
treatment and is involved with removal of some glycoproteins and 
proteoglycans from the collagenous matrix. Acid solutions known in the art 
may be used and may include but are not limited to hydrochloric acid 
(HCl), acetic acid (CH.sub.3 COOH) and sulfuric acid (H.sub.2 SO.sub.4). A 
preferred acid is hydrochloric acid (HCl) at a concentration preferably 
between about 0.5 to about 2 M, more preferably between about 0.75 to 
about 1.25 M; most preferably around 1 M. The final pH of the acid/salt 
solution is preferably between about 0 to about 1, more preferably between 
about 0 and 0.75, and most preferably between about 0.1 to about 0.5. 
Hydrochloric acid and other strong acids are most effective for breaking 
up nucleic acid molecules while weaker acids are less effective. Salts 
that may be used are preferably inorganic salts and include but are not 
limited to chloride salts such as sodium chloride (NaCl), calcium chloride 
(CaCl.sub.2), and potassium chloride (KCl) while other effective salts may 
be determined by one of skill in the art. Preferably chloride salts are 
used at a concentration preferably between about 0.1 to about 2 M; more 
preferably between about 0.75 to about 1.25 M; most preferably around 1 M. 
A preferred chloride salt for use in the method is sodium chloride (NaCl). 
In the most preferred embodiment, the tissue is contacted with 1 M HCl/1 M 
NaCl in water. The tissue is contacted preferably by immersion in the 
acid/salt solution while effective treatment is obtained by agitation of 
the tissue and the solution together for a time for the treatment step to 
be effective. 
The tissue is then contacted with a effective amount of salt solution which 
is preferably buffered to about a physiological pH. The buffered salt 
solution neutralizes the material while reducing swelling. Salts that may 
be used are preferably inorganic salts and include but are not limited to 
chloride salts such as sodium chloride (NaCl), calcium chloride 
(CaCl.sub.2), and potassium chloride (KCl); and nitrogenous salts such as 
ammonium sulfate (NH.sub.3 SO.sub.4) while other effective salts may be 
determined by one of skill in the art. Preferably chloride salts are used 
at a concentration preferably between about 0.1 to about 2 M; more 
preferably between about 0.75 to about 1.25 M; most preferably about 1 M. 
A preferred chloride salt for use in the method is sodium chloride (NaCl). 
Buffering agents are known in the art and include but are not limited to 
phosphate and borate solutions while others can be determined by the 
skilled artisan for use in the method. One preferred method to buffer the 
salt solution is to add phosphate buffered saline (PBS) preferably wherein 
the phosphate is at a concentration from about 0.001 to about 0.02 M and a 
salt concentration from about 0.07 to about 0.3 M to the salt solution. A 
preferred pH for the solution is between about 5 to about 9, more 
preferably between about 7 to about 8, most preferably between about 7.4 
to about 7.6. In the most preferred embodiment, the tissue is contacted 
with 1 M sodium chloride (NaCl)10 mM phosphate buffered saline (PBS) at a 
pH of between about 7.0 to about 7.6. The tissue is contacted preferably 
by immersion in the buffered salt solution while effective treatment is 
obtained by agitation of the tissue and the solution together for a time 
for the treatment step to be effective. 
After chemical cleaning treatment, the tissue is then preferably rinsed 
free of chemical cleaning agents by contacting it with an effective amount 
of rinse agent Agents such as water, isotonic saline solutions and 
physiological pH buffered solutions can be used and are contacted with the 
tissue for a time sufficient to remove the cleaning agents. A preferred 
rinse solution is physiological pH buffered saline such as phosphate 
buffered saline (PBS). Other means for rinsing the tissue of chemical 
cleaning agents can be determined by one of skill in the art. The cleaning 
steps of contacting the tissue with an alkaline chelating agent and 
contacting the tissue with a acid solution containing salt may be 
performed in either order to achieve substantially the same cleaning 
effect. The solutions may not be combined and performed as a single step, 
however. 
A preferred composition of the invention is a chemically cleaned tissue 
matrix derived from native, normally cellular tissues. The cleaned tissue 
matrix is essentially acellular telopeptide collagen, about 93% by weight, 
with less than about 5% glycoproteins, glycosaminoglycans, proteoglycans, 
lipids, non-collagenous proteins and nucleic acids such as DNA and RNA. 
Importantly, the bioremodelability of the tissue matrix is preserved as it 
is free of bound detergent residues that would adversely affect the 
bioremodelability of the collagen. Additionally, the collagen molecules 
have retained their telopeptide regions as the tissue has not undergone 
treatment with enzymes during the cleaning process. 
Tissue matrices are derived from dermis, artery, vein, pericardium, heart 
valves, dura mater, ligaments, intestine and fascia. A most preferred 
composition is a chemically cleaned intestinal collagen layer derived from 
the small intestine. Suitable sources for small intestine are mammalian 
organisms such as human, cow, pig, sheep, dog, goat or horse while small 
intestine of pig is the preferred source. In one preferred embodiment, the 
collagen layer comprises the tunica submucosa derived from porcine small 
intestine. In another embodiment, the collagen layer comprises the tunica 
submucosa and the basilar layers of the small intestine. The basilar 
layers consist of lamina muscularis mucosa and, if present in the native 
tissue, the stratum compactum. 
The most preferred composition of the invention is the intestinal collagen 
layer, cleaned by the chemical cleaning method of the invention, is 
essentially collagen, primarily Type I collagen, with less than about 5% 
glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous 
proteins and nucleic acids such as DNA and RNA. The collagen layer is free 
of bound detergent residues that would adversely affect the 
bioremodelability of the collagen. The collagen layer is substantially 
free of cells and cellular debris, including endogenous nucleic acids such 
as DNA and RNA and lipids. Further, the intestinal collagen layer is both 
sterile and endotoxin free when processed using sterile equipment, 
solutions and aseptic technique. 
Once the collagenous tissue matrix has been rendered substantially 
acellular and free of substantially noncollagenous extracellular matrix 
components, prostheses for implantation or engraftment may be manufactured 
therefrom. Collagen layers may be sutured or bonded together by use of any 
variety of techniques known in the art Methods for bonding the layers may 
employ adhesives such as thrombin, fibrin or synthetic materials such as 
cyanomethacrylates or chemical crosslinking agents. Other methods may 
employ heat generated by laser, light, or microwaves. Convection ovens and 
heated liquid baths may also be employed. 
Thermal welding of the collagen layers is the preferred method for bonding 
together the collagen layers of the invention. Methods for thermal welding 
of collagen are described in WO 95/22301, WO 96131157 and U.S. Pat. No. 
5,571,216, the teachings of which are incorporated herein by reference. 
The ICL is first cut longitudinally and flattened onto a solid, flat 
plate. One or more successive layers are then superimposed onto one 
another, preferably in alternating perpendicular orientation. A second 
solid flat plate is placed on top of the layers and the two plates are 
clamped tightly together. The complete apparatus, clamped plates and 
collagen layers, are then heated for a time and under conditions 
sufficient to effect the bonding of the collagen layers together. The 
amount of heat applied should be sufficiently high to allow the collagen 
to bond, but not so high as to cause the collagen to irreversibly 
denature. The time of the heating and bonding will depend upon the type of 
collagen material layer used, the moisture content and thickness of the 
material, and the applied heat. A typical range of heat is from about 
50.degree. C. to about 75.degree. C., more typically 60.infin.C. to 
65.degree. C. and most typically 62.infin.C. A typical range of times will 
be from about 7 minutes to about 24 hours, typically about one hour. The 
degree of heat and the amount of time that the heat is applied can be 
readily ascertained through routine experimentation by varying the heat 
and time parameters. The bonding step may be accomplished in a 
conventional oven, although other apparatus or heat applications may be 
used including, but not limited to, a water bath, laser energy, or 
electrical heat conduction. Immediately following the heating and bonding, 
the collagen layers are cooled, in air or a water bath, at a range between 
room temperature at 20.infin.C. and 1.infin.C. Rapid cooling, termed 
quenching, is required to stop the heating action and to create an 
effective bond between the collagen layers. To accomplish this step, the 
collagen layers may be cooled, typically in a water bath, with a 
temperature preferably between about 1.degree. C. to about 10.degree. C., 
most preferably about 4.degree. C. Although cooling temperatures below 
1.degree. C. may be used, care will need to be taken not to freeze the 
collagen layers, which may cause structural damage. In addition, 
temperatures above 10.degree. C. may be used in quenching, but if the 
temperature of the quench is too high, then the rate of cooling may not be 
sufficient to fix the collagen layers to one another. 
In the preferred embodiment, the collagenous material is crosslinked. 
Crosslinking imparts increased strength and structural integrity to the 
formed prosthetic construct while regulating the bioremodeling of the 
collagen by cells when the construct is implanted into a patient. Collagen 
crosslinking agents include glutaraldehyde, formaldehyde, carbodiimides, 
hexamethylene diisocyanate, bisimidates, glyoxal, adipyl chloride, 
dialdehyde starch, and certain polyepoxy compounds such as glycol 
diglycidyl ether, polyol polyglycidyl ether and dicarboxylic acid 
diglycidylester. Dehydrothermal, UV irradiation and/or sugar-mediated 
methods may also be used. Collagen will also naturally crosslink with age 
standing at room temperature. However, crosslinking agents need not be 
limited to these examples as other crosslinking agents and methods known 
to those skilled in the art may be used. Crosslinking agents should be 
selected so as to produce a biocompatible material capable of being 
remodeled by host cells. A preferred crosslinking agent is 
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The 
crosslinking solution containing EDC and water may also contain acetone. 
Crosslinking with EDC had been described in International PCT Publication 
Nos. WO 95/22301 and WO 96/31157. 
In some embodiments, additional collagenous layers may be added to either 
the outer or inner surfaces of the bonded collagen layers, either before 
or after crosslinking. In tubular constructs, as in a vascular construct, 
dense fibrillar collagen may be added to the luminal surface to create a 
smooth flow surface for its ultimate application as described in 
International PCT Publication No. WO 95/22301, incorporated herein by 
reference. This smooth collagenous layer also promotes host cell 
attachment, as in the formation of neointima, which facilitates ingrowth 
and bioremodeling of the construct. As described in International PCT 
Publication No. WO 95/22301, this smooth collagenous layer may be made 
from acid-extracted fibrillar or non-fibrillar collagen, which is 
predominantly type I collagen, but may also include other types of 
collagen. The collagen used may be derived from any number of mammalian 
sources, typically bovine, porcine, or ovine skin or tendons. The collagen 
preferably has been processed by acid extraction to result in a fibril 
dispersion or gel of high purity. Collagen may be acid-extracted from the 
collagen source using a weak acid, such as acetic, citric, or formic acid. 
Once extracted into solution, the collagen can be salt-precipitated using 
NaCl and recovered, using standard techniques such as centrifugation or 
filtration. Details of acid extracted collagen from bovine tendon are 
described, for example, in U.S. Pat. No. 5,106,949, incorporated herein by 
reference. 
Heparin can be applied to the prosthesis, by a variety of well-known 
techniques. For illustration, heparin can be applied to the prosthesis in 
the following three ways. First, benzalkonium heparin (BA-Hep) solution 
can be applied to the prosthesis by dipping the prosthesis in the solution 
and then air-drying it. This procedure treats the collagen with an 
ionically bound BA-Hep complex. Second, EDC can be used to activate the 
heparin, then to covalently bond the heparin to the collagen fiber. Third, 
EDC can be used to activate the collagen, then covalently bond protamine 
to the collagen and then ionically bond heparin to the protamine. Many 
other coating, bonding, and attachment procedures are well known in the 
art which could also be used. 
Treatment of the tissue matrix material with agents such as growth factors 
or pharmaceuticals in addition to or in substitution for heparin may be 
accomplished. The agents may include for example, growth factors to 
promote vascularization and epithelialization, such as macrophage derived 
growth factor (MDGF), platelet derived growth factor (PDGF), vascular 
endothelial cell derived growth factor (VEGF); antibiotics to fight any 
potential infection from the surgery implant; or nerve growth factors 
incorporated into the inner collagenous layer when the prosthesis is used 
as a conduit for nerve regeneration. In addition to or in substitution for 
drugs, matrix components such as proteoglycans or glycoproteins or 
glycosaminoglycans may be included within the construct. 
The collagenous prosthesis thus formed can also be sterilized in a dilute 
peracetic acid solution with a neutral pH. Methods for sterilizing 
collagen are described U.S. Pat. No. 5,460,962 and are incorporated by 
reference herein. In the preferred method, the collagen is disinfected 
with a dilute peracetic acid solution at a neutral pH. The peracetic acid 
concentration is preferably between about 0.01 and 0.3% v/v in water at a 
neutralized pH between about pH 6 and pH 8. Alternatively, sterilization 
with gamma irradiation, at typically 2.5 Mrad, or with gas plasma can also 
be used to sterilize the collagen. Other methods known in the art for 
sterilizing collagen may also be used. 
The following examples are provided to better explain the practice of the 
present invention and should not be interpreted in any way to limit the 
scope of the present invention. Those skilled in the art will recognize 
that various modifications can be made to the methods described herein 
while not departing from the spirit and scope of the present invention. 
EXAMPLES 
Example 1 
Chemical cleaning of mechanically stripped porcine small intestine 
The small intestine of a pig was harvested and mechanically stripped, using 
a Bitterling gut cleaning machine (Nottingham, UK) which forcibly removes 
the fat, muscle and mucosal layers from the tunica submucosa using a 
combination of mechanical action and washing using water. The mechanical 
action can be described as a series of rollers that compress and strip 
away the successive layers from the tunica submucosa when the intact 
intestine is run between them. The tunica submucosa of the small intestine 
is comparatively harder and stiffer than the surrounding tissue, and the 
rollers squeeze the softer components from the submucosa. The result of 
the machine cleaning was such that the submucosal layer of the intestine 
solely remained. The remainder of the procedure was performed under 
aseptic conditions and at room temperature. The chemical solutions were 
all used at room temperature. The intestine was then cut lengthwise down 
the lumen and then cut into 15 cm sections. Material was weighed and 
placed into containers at a ratio of about 100:1 v/v of solution to 
intestinal material. 
A. To each container containing intestine was added approximately 1 L 
solution of 0.22 mm (micron) filter sterilized 100 mM 
ethylenediaminetetraacetic tetrasodium salt (EDTA)/10 mM sodium hydroxide 
(NaOH) solution. Containers were then placed on a shaker table for about 
18 hours at about 200 rpm. After shaking, the EDTA/NaOH solution was 
removed from each bottle. 
B. To each container was then added approximately 1 L solution of 0.22 mm 
filter sterilized 1 M hydrochloric acid (HCl)/1 M sodium chloride (NaCl) 
solution. Containers were then placed on a shaker table for between about 
6 to 8 hours at about 200 rpm. After shaking, the HCl/NaCl solution was 
removed from each container. 
C. To each container was then added approximately 1 L solution of 0.22 mm 
filter sterilized 1 M sodium chloride (NaCl)/10 mM phosphate buffered 
saline (PBS). Containers were then placed on a shaker table for 
approximately 18 hours at 200 rpm. After shaking, the NaCl/PBS solution 
was removed from each container. 
D. To each container was then added approximately 1 L solution of 0.22 mm 
filter sterilized 10 mM PBS. Containers were then placed on a shaker table 
for about two hours at 200 rpm. After shaking, the phosphate buffered 
saline was then removed from each container. 
E. Finally, to each container was then added approximately 1 L of 0.22 mm 
filter sterilized water. Containers were then placed on a shaker table for 
about one hour at 200 rpm. After shaking, the water was then removed from 
each container. 
Treated samples were cut and fixed for histological analyses. Hemotoxylin 
and eosin (H&E) and Masson trichrome staining was performed on both 
cross-section and long-section samples of both control and treated 
tissues. Treated tissue samples appeared free of cells and cellular debris 
while control samples appeared normally and expectedly very cellular. 
Example 2 
Chemical Cleaning of Porcine Heart Valve 
A porcine heart was procured from a 1 pound piglet and shipped in 
physiological pH saline on ice. Within 4 hours, the heart valves were 
removed from the heart mass using scalpel and forceps. Some further gross 
dissection was performed to remove excess tissue from around the valves. 
One valve was retained as a control with sample pieces cut and fixed for 
various histological analyses while the other valve underwent the chemical 
cleaning process. The remainder of the procedure was performed under 
aseptic conditions and at room temperature. The chemical solutions were 
all used at room temperature. 
The valve was placed into 1 L solution of 100 mM EDTA/10 mM NaOH for about 
18 hours while agitating on a shaker platform. The valve was then placed 
into 1 L of 1 M HCl/1 M NaCl and agitated for 8 hours. The valve was then 
placed into 1 L solution of 1 M HCl/10 mM phosphate buffered saline (PBS) 
and agitated for about 18 hours. The valve was then rinsed in PBS for 
between about 2-4 hours and then finally rinsed in sterile water for about 
1 hour while agitating. Treated sample pieces were then cut and fixed for 
various histological analyses. 
Hemotoxylin and eosin (H&E) and Masson trichrome staining was performed on 
both cross-section and long-section samples of both control and treated 
valves. Treated valve samples appeared free of cells and cellular debris 
while control samples appeared normally and expectedly very cellular. 
Example 3 
Chemical Cleaning of Porcine Artery, Pericardium and Fascia 
A segment of femoral artery, the entire pericardium, and fascia were 
procured from a 450 lb. sow. The tissues were shipped in physiological pH 
saline on ice. The tissues were dissected further to remove excess tissue. 
Samples of each tissue were taken without cleaning for control samples and 
fixed for various histological analyses while the remainder of the tissues 
underwent the chemical cleaning process. The remainder of the procedure 
was performed under aseptic conditions and at room temperature. The 
chemical solutions were all used at room temperature. 
The tissues were separately placed into 1 L solution of 100 mM EDTA/10 mM 
and agitated on a shaker platform for about 18 hours. The tissues were 
then each separately placed into 1 L solution of 1 M HCl/1 M NaCl and 
agitated for 8 hours. Next, the tissues were separately placed into a 1 L 
solution of 1 M HCl/10 mM phosphate buffered saline (PBS) and then 
agitated for about 18 hours. The tissues were then separately rinsed in 
PBS for between about 2 to 4 hours and then finally rinsed in sterile 
water for about 1 hour while agitating. Treated sample pieces were then 
cut and fixed for various histological analyses. 
Hemotoxylin and eosin (H&E) and Masson trichrome staining was performed on 
both cross-section and long-section samples of both control and treated 
tissues. Treated tissue samples appeared free of cells and cellular debris 
while control samples appeared normally and expectedly very cellular. 
Example 4 
Differently Ordered Chemical Cleaning 
This procedure was performed under aseptic conditions and at room 
temperature and all chemical solutions were used at room temperature. 
Mechanically stripped porcine intestine was cut into five 15 cm sections as 
described in example 1. 
To each container was then added approximately 1 L of 0.22 mm filter 
sterilized solution of 1 M hydrochloric acid (HCl)/1 M sodium chloride 
(NaCl). Containers were then placed on a shaker table for between about 6 
to 8 hours at about 200 rpm. After shaking, the HCl/NaCl solution was 
removed from each container. 
To each container containing intestine was added approximately 1 L of 0.22 
mm (micron) filter sterilized solution of 100 mM 
ethylenediaminetetraacetic (EDTA)/10 mM sodium hydroxide (NaOH) solution. 
Containers were then placed on a shaker table for about 18 hours at about 
200 rpm. After shaking, the EDTA/NaOH solution was removed from each 
bottle. 
To each container was then added approximately 1 L of 0.22 mm filter 
sterilized solution of 1 M sodium chloride (NaCl)/10 mM phosphate buffered 
saline (PBS). Containers were then placed on a shaker table for 
approximately 18 hours at 200 rpm. After shaking, NaCl/PBS solution was 
removed from each container. 
To each container was then added approximately 1 L of 0.22 mm filter 
sterilized solution of 10 mM PBS. Containers were then placed on a shaker 
table for about one hour at 200 rpm. After shaking, the phosphate buffered 
saline was then removed from each container. 
Finally, to each container was then added approximately 1 L of 0.22 mm 
filter sterilized water. Containers were then placed on a shaker table for 
about one hour at 200 rpm. After shaking, the water was then removed from 
each container. 
Treated sample pieces were then cut and fixed for various histological 
analyses. Hemotoxylin and eosin (H&E) and Masson trichrome staining was 
performed on both cross-section and long-section samples of both control 
and treated tissues. Treated tissue samples appeared free of cells and 
cellular debris while control samples appeared normally and expectedly 
very cellular. 
Example 5 
Various alkaline and chelating agents 
The cleaning of mechanically stripped porcine intestinal submucosa was 
followed as according to example 1. This procedure was performed under 
aseptic conditions and at room temperature and all chemical solutions were 
used at room temperature. The chemical cleaning process of example 1 was 
followed but with the substitution of the alkaline chelating agent of step 
A was substituted for other alkaline chelating agents of similar nature: 
A. To each container containing intestine was added approximately 1 L of 
0.22 mm (micron) filter sterilized solution of either 100 mM 
ethylenebis(oxyethylenitrilo)tetraacetic acid (EGTA)/10 mM NaOH; 100 mM 
EDTA/10 mM Ca(OH)2 (calcium hydroxide); or, 100 mM EDTA/10 mM K2CO3 
(potassium carbonate) solution. Containers were then placed on a shaker 
table for about 18 hours at about 200 rpm. After shaking, the alkaline 
chelating agents solution was removed from each bottle. 
B. To each container was then added approximately 1 L of 0.22 mm filter 
sterilized solution of 1 M hydrochloric acid (HCl)/1 M sodium chloride 
(NaCl) solution. Containers were then placed on a shaker table for between 
about 6 to 8 hours at about 200 rpm. After shaking, the HCl/NaCl solution 
was removed from each container. 
C. To each container was then added approximately 1 L of 0.22 mm filter 
sterilized solution of 1 M sodium chloride (NaCl)/10 mM phosphate buffered 
saline (PBS). Containers were then placed on a shaker table for 
approximately 18 hours at 200 rpm. After shaking, NaCl/PBS solution was 
removed from each container. 
D. To each container was then added approximately 1 of 0.22 mm filter 
sterilized solution of 10 mM PBS. Containers were then placed on a shaker 
table for about one hour at 200 rpm. After shaking, the phosphate buffered 
saline was then removed from each container. 
E. Finally, to each container was then added approximately 1 of 0.22 mm 
filter sterilized water. Containers were then placed on a shaker table for 
about one hour at 200 rpm. After shaking, the water was then removed from 
each container. Samples were fixed for histological analyses. 
Hemotoxylin and eosin (H&E) and Masson trichrome staining was performed on 
both cross-section and long-section samples of both control and treated 
tissues. Treated tissue samples appeared free of cells and cellular debris 
while control samples appeared normally and expectedly very cellular. 
Example 6 
Various acid and salt agents 
The mechanically stripped porcine intestinal submucosa of example 1 was 
chemically cleaned using a substituted acid agent or substituted salt 
agent in step B. This procedure was performed under aseptic conditions and 
at room temperature and all chemical solutions were used at room 
temperature. 
A. To each container containing intestine was added approximately 1 L 
solution of 0.22 mm (micron) filter sterilized 100 mM 
ethylenediaminetetraacetic tetrasodium salt (EDTA)/10 mM sodium hydroxide 
(NaOH) solution. Containers were then placed on a shaker table for about 
18 hours at about 200 rpm. After shaking, the EDTA/NaOH solution was 
removed from each bottle. 
B. To each container was then added approximately 1 L of 0.22 mm filter 
sterilized solution of either 1 M CH3COOH (acetic acid)/1 M NaCl or 1 M 
H2SO4 (sulfuric acid)/1 M NaCl solution. Containers were then placed on a 
shaker table for between about 6 to 8 hours at about 200 rpm. After 
shaking, the solution was removed from each container. 
C. To each container was then added approximately 1 L of 0.22 mm filter 
sterilized 1 M sodium chloride (NaCl)/10 mM phosphate buffered saline 
(PBS). Containers were then placed on a shaker table for approximately 18 
hours at 200 rpm. After shaking, NaCl/PBS solution was removed from each 
container. 
D. To each container was then added approximately 1 L of 0.22 mm filter 
sterilized 10 mM PBS. Containers were then placed on a shaker table for 
about one hour at 200 rpm. After shaking, the phosphate buffered saline 
was then removed from each container. 
E. Finally, to each container was added approximately 1 L of 0.22 mm filter 
sterilized water. Containers were then placed on a shaker table for about 
one hour at 200 rpm. After shaking, the water was then removed from each 
container. 
Treated sample pieces were then cut and fixed for various histological 
analyses. Hemotoxylin and eosin (H&E) and Masson trichrome staining was 
performed on both cross-section and long-section samples of both control 
and treated tissues. Treated tissue samples appeared free of cells and 
cellular debris while control samples appeared normally and expectedly 
very cellular. 
Example 7 
Glycosaminoglycan (GAG) Content of ICL Determined by Cellulose 
Acetate Gel Electrophoresis and Alcian Blue Assay 
To determine GAG content of ICL, cellulose acetate gel electrophoresis with 
subsequent alcian blue stain was performed on extracts of chemically 
cleaned ICL. 
Samples of ICL underwent the chemical cleaning regimen outlined in Example 
1, cut into 0.125 cm2 pieces and placed into eppendorf tubes. To digest 
the samples, 100 .mu.l of papain (0.1 mg/ml papain in 0.1 M sodium 
phosphate, 0.1 M sodium chloride, 0.005 M EDTA, 0.9 mg/ml cysteine, pH 
5.8) was added to each tube and allowed to incubate for about 18 hours at 
60.degree. C. Standard containing known amounts of GAG (heparin) were 
prepared in parallel. Dowex (0.4 g HCl form) and 3 ml water were then 
added. After spinning to remove the Dowex resin, 1 ml was removed and 
lyophilized. The samples were then rehydrated in 100 .mu.l purified water 
and centrifuged for about 5 minutes. 
Samples were separated on cellulose-acetate sheets using the method of 
Newton, et al. (1974). Cellulose-acetate sheets were soaked in 0.1 M 
lithium chloride/EDTA buffer (pH 5.8) and blotted gently. Samples (5 .mu.l 
each) were applied to the sheets at the cathode end and electrophoresed 
for 30 minutes at 5 mA. 
Following electrophoresis, the sheets were immersed immediately in an 
alcian blue stain solution (0.2% alcian blue 8GX, 0.05 M magnesium 
chloride, 0.025 M sodium acetate buffer (pH 5.8) in 50% ethylene alcohol) 
and placed on a shaker platform for about 30 minutes at room temperature. 
The sheets were then destained in at least three washes of destaining 
solution (0.05 M magnesium chloride, 0.025 M sodium acetate buffer (pH 
5.8) in 50% ethylene alcohol) for a total of about 30 minutes on a shaker 
platform. No detectable GAG staining was observed for papain digested ICL 
while as little as 0.005 microgram heparin standard was detectable. 
These results showed that the total amount of GAG remaining in chemically 
cleaned ICL is less than 1% (dry weight). 
Example 8 
Lipid Content of ICL Determined by Methylene Chloride Extraction 
ICL was laid out flat on plastic plates and air dried for two hours. Once 
dried, ICL was cut into smaller pieces of about 1 cm2 of which 1.100 g 
were transferred to a soxhlet thimble. 
To a Kontes brand round bottom flask 24/40 was added 90 ml methylene 
chloride. The soxhlet was assembled in the fume hood with the bottom of 
the flask in a heated water bath and ice cooled water running through the 
distiller. 
Extraction was allowed to proceed for four hours after which the soxhlet 
was disassembled. The round bottom flask containing the solvent and 
extracted material was left in the heated water bath until methylene 
chloride was evaporated until there remained 5 ml. The methylene chloride 
was then transferred to a 11.times.13 glass culture tube and the remaining 
solvent was boiled off. To the tube was added 2 ml of methylene chloride 
and the tube was capped immediately and the tube placed in a -20.degree. 
C. freezer. 
The weight of the extracted material was then determined. The glass tube 
was placed in an ice bath. The weight of a Ludiag 1.12 ml aluminum weigh 
boat was tared on a microbalance (Spectrum Supermicro). 10 .mu.l of 
resuspended extraction was added to the weigh boat and the solvent was 
boiled off by placing the weigh boat on a hot plate for 45 seconds. The 
weigh boat was allowed to cool for about 190 seconds and was placed on the 
microbalance. The procedure was then repeated for extract volumes of 20 
.mu.l and 30 .mu.l. 
Results indicate that the percentage of lipid is less than about 0.7% lipid 
by weight in dry chemically cleaned ICL. In contrast, non-chemically 
cleaned ICL contains a higher fraction of lipid; at least about 1.5% by 
weight in dry ICL that has not been chemically cleaned by the method of 
the invention. 
Example 9 
Amino Acid Analysis of ICL 
Collagens are proteins characterized by their triple-helical regions which 
have a repeating triplet of amino acids glycine-X-Y, where X is frequently 
proline and Y is often hydroxyproline. Hydroxyproline is frequently used 
as an amino acid to identify and quantify collagens. Udenfriend, Science, 
152:1335-1340 (1966). 
To determine complete amino acid analysis of ICL, PICO-TAG HPLC was 
performed on mechanically cleaned (not chemically cleaned) porcine ICL and 
chemically cleaned ICL. Hydroxyproline content was measured for both 
materials and compared. 
Sample pieces of ICL from each condition weighing about from 0.31 to about 
0.36 g were dried further using a CEM AVC80 oven (CEM Corp.; Matthews, 
N.C.). Smaller samples were cut from these dried ICL pieces weighing about 
9.5 to about 13.1 mg. Samples were placed into screw cap culture tubes and 
the samples were then hydrolyzed (n=3 for each condition) in 1% phenol in 
6 M HCl at 110.degree. C. for about 16 hours. ICL hydrolysates were then 
diluted in 0.1 M HCl to normalize the material concentrations to 1 mg/ml. 
To labeled glass tubes (6.times.55), 20 ml of hydrolysates and 8 ml of 
1.25 mmol/ml L-norleucine as an internal standard. Samples were then 
frozen and lyophilized. Samples were then re-dried by adding 20 ml of 
2:2:1 ethanol:water:triethylamine to the tubes, freezing and lyophilizing. 
Samples were then derivatized for 20 minutes at room temperature by adding 
20 ml of reagent (7:1:1:1 ethanol:water:triethylamine:PITC) followed by 
freezing and lyophilizing. Samples were finally suspended in 200 ml 
PICO-TAG Sample Diluent and aliquoted to HPLC vials. 
Amino acid standards were prepared in the following manner: 0.1 ml of amino 
acid standard (Product #: A-9531, Sigma) was added to 1.9 ml 0.1 M HCl. 
Five serial dilutions at 1:1 were made using 0.1 M HCl. Volumes of 100 ml 
for each serial dilution and 8 ml of 1.25 mmol/ml L-norleucine were 
together added to glass tubes (6.times.55) and then prepared in the same 
manner as ICL samples. 
Samples and standards were run on a 3.9.times.150 mm PICO-TAG Amino Acid 
column (Part# 88131; Waters Corp.; Milford, Mass.). Injections of 10 ml 
for samples and 20 ml for standards were analyzed in triplicate for each. 
Results indicate for chemically cleaned ICL material, the content of major 
collagenous amino acids in the material approach that of purified collagen 
preparations. Using the hydroxyproline as a measure of collagen content, 
the percentage of collagen by weight in ICL is calculated to be at least 
about 93% collagen by weight. In contrast, non-chemically cleaned ICL 
contains a high fraction of non-collagenous amino acids; between about 11 
to 25% by weight of ICL is non-collagenous material. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity and understanding, it 
will be obvious to one of skill in the art that certain changes and 
modifications may be practiced within the scope of the appended claims.