CA2277896C - Preparation of collagen - Google Patents

Abstract

The present invention relates to methods for preparing collagen, especially type I collagen. In particular, the present invention provides methods for the preparation of collagen suitable for biomedical and veterinary applications.
The collagen prepared according to the present invention provides numerous desirable characteristics for applications such as implantation, transplantation, and grafting.

Description

PREPARATION OF COLLAGEN
FIELD OF THE INVENTION
The present invention relates to methods for preparing collagen ti-om tissues of humans and other animals. In particular, the present invention provides methods for the preparation of collaLen suitable for biomedical, veterinary. and other applications.
BACKGROUND OF THE INVENTION
Collagen is the most abundant protein in mammals. (.See~, U.S. Patem No.
5,043.426 to Goldstein). Indeed, it represents 30% of the dry weight of the human body. (,See, L.C. Junqueira and J. Carneiro, Baric Hi~lolu~~, 4th ed.. Lange Medical Publications. Los Altos, CA, [1983], pp. 89-119). Vertebrate collagen is actually a family of proteins produced by several cell types. Within this protein family, the collagen types are distinguishable by their chemical compositions. different morphological and I S pathological features, distributions within tissues, and their functions.
Although many types of collagen have been described, five major types have been recognized.
A. Forms Of Collagen Collagen type I is the most abundant form of collagen. with widespread distribution within the body. It is present in tissues in structures classically referred to as "collagen titers" that form bones, dentin, tendons, fascias, sclera, organ capsules.
dertnis, fibrous cartilage, etc. The primary function of type I collagen is to resist tension.
Microscopically.
type I collagen appears as closely packed, thick, non-argyrophilic, strongly birefringent red or yellow titers. Its ultrastructure is characterized as being densely packed, thick fibrils with marked variation in diameter. It is produced by fibroblasts, osteoblasts.
odontoblasts, and chondroblasts.
Collagen type II is primarily found in cartilage (c~.g., hyaline and elastic cartilages).
The primary function of type II collagen is to resist intermittent pressure.
Microscopically, it appears as a loose, collagenous network, that is visible only with picrosirius stain and polarization microscopy. Ultrastructurally, it is characterized as appearing to have no fibers, but with very thin fibrils embedded in abundant gro!and substance. It is produced by chondrobiasts.

C'olla~Ten type III is commonly associated with type I collagen in tissues.
and may be the colla~~enous component of reticular fibers. It is present in smooth muscles. en doneurium, arteries. uterus. liver, spleen. kidney. an lung tissue. The primary function of type III
collagen is to maintain the structure of expansible organs. Microscopically.
it appears as a loose network of thin, argyrophilic. and weakly birefringent greenish fibers.
Ultrastructurally.
it is characterized as beings loosely packed thin fibrils with fairly uniform diameters. (t is produced by SI1100Ih IIIIISCIc fibroblasts. reticular cells, Schwann cells.
andhepatocytes.
Collagen type IV is found in the epithelial and endothelial basal lamina and basement membranes. The primary function of type IV collagen involves support and filtration.
Microscopically. it appears as a thin, amorphous. weakly birefringent membrane.
Ultrastructurally, it appears to have neither fibers nor fibrils.
Collagen type V is found in fetal membranes, blood vessels, placental basement membrane. and in small amounts in other tissues. This type of collagen remains largely uncharacterized.
1~
B. Structure Of Collagen The principal amino acids found in collagen are glycine, proline and hydroxyproline.
Hydroxylysine is also characteristic of collagen. These hydroxy amino acids are the result of hydroxylation of proline and lysine present in nascent collagen polypeptides during collagen ?0 synthesis. The collagen content in a tissue can be determined by measurement of its hydroxyproline content.
Collagen is comprised of polypeptide chains. designated as "a." There are two types of a chains, referred to as "alpha- I " ("a 1 ") and "alpha-2 ( "a -2"). The most important types of a 1 chains are a 1 (I), a 1 (II), a 1 (III), and a 1 (IV), which aggregate in different 25 combinations to produce the triple helices of types I, II, III, IV, and V.
Type I collagen is composed of two a 1 and one a2 chains. It's formula is (a 1 [IJ), a 2. The formula for type II collagen is (al[II]);, while the formula for type III collagen is (aIjIII]);, and type IV is (cx 1 [IV]);.
"Tropocollagen" is the protein unit that polymerizes into al;gregations of microt76rillar 30 subunits packed together to form "collagen fibrils." Hydroten bonds and hydrophobic interactions are critical in this aggregation and packing. Covalent crosslinks reinforce the structure of the collagen fibrils. Collagen fibrils are thin and elongated. of variable diameter.
and have transverse striations with a characteristic periodicity of 64 nm. The transverse I

striations is produced by the overlapping organization of the subunit tropocollagen molecules.
In type 1 and III collagen. these fibrils associate to produce collagen "fibers." In collaLen ype I. colla~ien "bundles" may be formed by association of the fibers.
Collagen type II is observed as fibrils. but does not form fibers, while types IV and V do not form fibrils or f i bers.
Collagen fibers are the most abundant t3ber found in connective tissue. Their inelasticitv and molecular configuration provide collagen fibers with a tensile stren~ah that is ~~reater than steel. Thus. collagen provides a combination of flexibility and strength to the tissues in which it resides. In many parts of the body, collagen fibers are organized in parallel arrays to form collagen "bundles."
When fresh. collagen fibers appear as colorless strands, although when a large number of fibers are present. they cause the tissues in which they reside to be white (c~.~~., tendons and aponeurosest. The organization of the elongated tropocollaLen in the fibers cause them to be birefringent. Staining with certain acidic dyes (e.,~~., Sirius red) enhances this biretringency.
1 s As this increase in birefringency us only observed in oriented collagen structures. it is useful as a method to detect the presence of collagen in a tissue.
C. Properties And Uses Of Collagen There are many properties of collagen that make it an attractive substance for various 2U medical applications. such as implants, transplants, organ replacement, tissue equivalents.
vitreous replacements. plastic and cosmetic surgery, surgical suture, surgical dressings for wounds. burns. etc. (,See e~.~=., U.S. Patent Nos. ~;lOG,949. x.104,660.
~.081.1OG. 6.383,9;0, 4.485.095. 4.486.097. 4.639.716, 4,546,500, 4.409,332. 4,604.346, 4.835.IU2, 4,837.;79.
3.800.79?, ;.491.760. 3,113,568, 3,471,598, 2,202,666, and 3,157,5?4:
J.F. Prudden, Arch. Surg. 89:1046-1059 [ 1964]; and E. E.
Peacock eW 1. Ann. Surg., 161:238-247 [1965)). For example, by itself:
collagen is a relatively weak immunogen, at least partially due to masking of potential antigenic determinants within the collagen structure. Also, it is resistant to proteolysis due to its helical structure. In addition. it is a natural substance for cell adhesion and the major tensile load-3U bearing component of the musculoskeletal system. Thus, extensive efforts have been devoted to the production of collagen fibers and membranes suitable for use in medical, as well as veterinary applications.
_,_ Collagen has been used in the area of soft tissue augmentation. as a replacement for paraffin. petrolatum, vegetable oils. lanolin. bees wax, and silicone previously used. (.See e.y., U.S. Patent No. 5.002.071). However, problems' have been associated with the use of collagen in implants. As the non-collagenous proteins present in impure colla~~en preparations are more potent immunogens than the collagen, and can stimulate the inflammatory response. it is critical that highly pure collagen be used. If the inflammatory cascade is stimulated. the resorption of collagen occurs by the infiltrating inflammatory cells (c~.~,~., macrophages. and granulocytes) that contain collagenase. resulting in thee digestion of the collagen. In addition, collagen itself is chemotactic.
and becomes I () increasingly chemotactic as it is degraded into smaller peptide fragments. Also, there are concerns associated with the use of non-human collagen. For example, a repeatedly documented prohlem associated with the use of bovine collagen as a biomaterial is the consistent. chronic cellular inflammatory reaction that is evident following its implantation or use. 'this inflammation may result in residual scar tissue formation. adhesion formation.
I ~ , interference with healing of skin edges, pseudointirna formation, pseudodiaphragm formation, disruption of anastomoses, transient low grade fever, aneurysms, or other problems.
D. Preparation Of Collagen Collagen preparations are typically prepared from skin, tendons (e~. ~,~., bovine Achilles, 20 tail. and extensor tendons), hide or other animal parts, by procedures involving acid and/or enzyme extraction. Basically. collagen preparation methods involve purification of collagen by extraction with diluted organic acids, precipitation with salts, optional gelation and/or lyophilization, tangential filtration etc. After separating facia, fat and the impurities. the tissue is subjected to moderate digestion with proteolytic enzymes, such as pepsin. then the ?5 collagen is precipitated at a neutral pH. redissoived and the residual impurities precipitated at an acid pl-I. The tissue is then digested with a strong alkali and then exposed to acid to facilitated swellinb. The collagen fibers are then precipitated with salts or ornanic solvents, and dehydrating the collagen fibers. (See c~.~,>., U.S. Patent No. 5,028,695).
Eventually the extracted collagen can be converted into a finely divided 30 fibrous collagen by treating water-wet collagen with acetone to remove water, centrifuging to obtain the solid mass of collagen and deaggregating the collagen during drying. (.Se'c' e.,~~., U.S. Patent No. 4148664). The collagen preparation can then be brought back to a neutral pH and dried in the form of fibers.
Completely transparent.

WO 98130225 PCTlUS98/00371 physiological and hemocompatible gels. collagen films. and solutions can be prepared. These i-orms of colla~~en may then be used in the fabrication oi' contact lenses and implants.
One disadvantage of treatment with pepsin, is that the collagen preparation may be partially degraded (i.c~.. the extraction enzymes cleave the collagen molecule at the terminal J doll-Ile11Ca1 regions. w'111Ch cOntalil the inter-collagenous cross-linkages). Indeed. it has been bound that colla~~en extracted with pepsin results in preparations that are too weak for certain applications. especially those for which substantial .mechanical handling of the collagen preparation is required.
Solve acid treatments also have disadvantages. For example, the acid process described by Chvapil (M. Chvapil et ul.. Intl. Rev. Connective Tiss. Res.. 6:1-55 [1979]) involves acid solubilization of bovine tendon collagen to produce a collagen suspension. This suspension is then either dialyzed or precipitated in saline. resulting in an amorphous precipitate containing non-tibrillary denatured collagen. Collagen prepared according to this method is generally not directly suitable for medical purposes. as it lacks tensile strength in moist media and has little resistance against enzymatic degradation when applied to living tissue. 1n addition, denatured collagen or collagen that has undergone treatment to reform the physical and biological characteristics to approximate collagen in vivo is often not satisfactory.
It often lacks the mechanical properties required fox wet dressings, as it lacks the ih viv«
organized structure (i.c~..collagen fibers are not present in this artificial collagen).
?0 Thus, current methods for collagen preparation are unsatisfactory. Clearly, there is a need for the development of improved methods for the high volume production of high duality collagen suitable for use in medical treatment.
SUMMARY OF THE INVENTION
The present invention relates to methods for preparing collagen from humans and other animals. In particular. the present invention provides methods for the preparation of collagen suitable for biomedical applications.
The present invention provides numerous embodiments for the purification of collagen.
It is particularly preferred that the collagen purified according to one embodiment of the ;0 present invention be type 1 collagen.
In one embodiment. the present invention provides a method for purifying collagen comprising: providing a sample comprising collagen, first and second proteofytic enzyme preparations. and a reducing agent: exposing the collagen sample to the tlrst proteolytic _5_ enzyme preparation to produce a first collagen solution; exposing the first collagen solution to the reducin~~ agent to produce a second collagen solution; exposing the second collagen SOIlltloll t0 the second proteolytic enzyme preparation to produce purified collagen.
In one alternative embodiment, the method of the present invention further comprises the step of de-epithelializing the sample prior to exposing the sample to the first proteolytic enzyme preparation.
In a preferred embodiment, the first and/or second proteolytic enzyme preparation comprises an enzyme in the cysteine class. In a particularly preferred embodiment, the tirst and/or second proteolvtic enzyme preparation comprises papain.
In an alternate embodiment of the method of the present invention. the reducing agent is selected from the group CoIISIStlllg of sodium sulfide. dithiothreitol, glutathionine, arid sodium borohydride. (n a preferred embodiment, the reducing agent comprises sodium borohvdride.
In yet another embodiment. the method of the present invention comprises the further 1 S step of exposing the puritled collagen to a delipidation agent to produce dilapidated collagen.
In a preferred embodiment of this method, the delipidation agent comprises a mixture comprising chloroform and methanol.
In a particularly preferred embodiment, the method of the present invention further comprises the steps of compressing the delipidated collagen to produce compressed collagen;
?0 dehydrating the compressed collagen to produce dehydrated collagen; and dispersing and drying the dehydratin~~ collagen to torm collagen fibers. Thus, in this elnbodiment of the methods of the present invention. the collagen fibers are dried.
In another alternative embodiment, the method of the present im~ention comprises the step of exposing the delipidated collagen to a phosphorylating agent to produce 25 phosphorylated collagen. In a preferred embodiment, the phosphorylation agent is selected t-rom the ~~roup consisting of sodium trimetaphosphate, sodium hexametaphosphate. sodium ultraphosphate, sodium tetratnetaphosphate. phosphoric anhydride, and phosphoryl trichloride.
In a particularly preferred embodiment, the phosphorylation agent comprises sodium trimetaphosphate. In an alternatively preferred embodiment. the purified collagen comprises JU ~~OllaLl'.IlhIZnT~'1 In one embodiment, the present invention provides purified colla~~en purified by the steps of: providing a sample comprising collagen, first and second proteolytic enzyme preparations. and a reducing agent; exposing the collagen sample to the first proteolytic enzyme preparation to produce a first collagen solution; exposing the first collagen solution to the reducin~~ agent to produce a second collagen solution, exposing the second collagen solution to the second proteolytic enzyme preparation to produce purified collagen.
It is turther contemplated that the purified collagen be comprised of additional compounds. includin~~ but not limited to antimicrobials, antivirals. growth factors, anti-dehydration compound's. antiseptics. or other compounds suitable for biomedical and/or voter tnarv uses.
The present invention also provides an alternative embodiment comprising methods for production of biocompatible collagen. in which the method con prises:
providing a sample comprising collagen. first and second proteolytic enzyme preparations. a reducing agent. a delipidation agent, and a phosphorvlation agent: exposing the collagen sample to the first proteolytic enzyme preparation to produce a first collagen solution: exposing the first cullagen solution to the reducing agent to produce a second collagen solution; exposing the second collagen solution to the second proteolytic enzyme preparation to produce to produce a 1 s proteolyzed collagen solution; exposing the proteolyzed collagen solution to the delipidation agent to produce delipidated collagen: and exposing the delipidated collagen to the phosphorylation agent to produce phosphorylated collagen.
In an alternative embodiment. the method of the present invention further comprises the step of de-epithelializing the sample prior to exposing the sample to the tlrst proteolytic 2U enzyme preparation.
In a preferred embodiment, the first and/or second proteolytic enzyme preparation of the method comprises an enzyme in the cysteine class. In a particularly preferred embodiment. the first and/or second proteolytic enzyme preparation comprises papain.
In an alternate embodiment of the method of the present invention, the reducing agent is selected tcom the group consisting of sodium sulfide, dithiothreitol.
glutathionine, and sodium borohydride. In a preferred embodiment, the reducing agent comprises sodium borohvdride.
In an alternative embodiment. the delipidation agent comprises a mixture of chloroform and methanol. In yet another embodiment, the phosphorylation agent is selected 3() from the group consisting of sodium trimetaphosphate, sodium hexatnetaphosphate, sodium ultraphosphate. sodium tetrametaphosphate, phosphoric anhydride, and phosphoryl trichloride.
In a particularly preferred embodiment, the phosphorylation agent comprises sodium trimetaphosphate.
_7_ 1 n yet a further embodiment. the method of the present invention further comprises the steps of compressin g< the delipidated collagen to produce compressed collagen; dehydratin~~
the compressed collagen to produce dehydrated collagen; and dispersing and drying; the dehydrating__= collagen to form collagen fibers. Thus, in this embodiment of the methods of the present invention, the collagen t3bers are dried.
In a particularly preferred embodiment, the method further comprises the step of tilter-sterilizin>r the ctelipidated colla~~en prior to exposing; the delipidated colla~~en to the phosphorvlation absent to produce phosphorylated collagen.
The present invention also provides purified such that the collagen is biocompatible.
In this embodiment. the biocompatible collagen is produced by the method of:
providing; a sample comprising collagen, first and second proteolytic enzyme preparations, a reducing atent. a delipidation agent, and a phosphorylation agent; exposing the collagen sample to the first proteolvtic enzyme preparation to produce a first collagen solution:
e~cposing the first collagen solution to the reducing agent to produce a second collagen solution;
exposing the second collagen solution to the second proteolytic enzyme preparation to produce to produce a proteolyzed collagen solution; exposing the proteolyzed collagen solution to the delipidation agent to produce delipidated collagen; and exposing; the delipidated collagen to the phosphorylation agent to produce phosphorylated collagen. In one preferred embodiment. the phosphorylated collagen comprises CollagenPROTM.
In another preferred embodiment, the biocompatible collagen comprises a film or membrane, while in alternate preferred embodiments, the biocompatible collagen comprises a solid. while in other alternately preferred embodiments, the biocompatible collagen comprises a solution. In other embodiments. the biocompatible collagen is a dried film that is hydrated prior to its application.
It is further contemplated that the biocompatible collagen be comprised of additional compounds. including) but not limited to antimicrobials, antivirals, growth factors, anti-dehydration compounds, antiseptics. or other compounds suitable for biomedical and/or veterinary uses.
The present invention also provides methods for achieving hemostasis comprising;:
providing purified and/or biocompatible collagen purified as described above, and a bleeding wound: and exposing; the purified and/or biocompatible collagen to the bleeding wound.
_g_ The present invention also provides a method of transplantation comprisin~~
providing purified and/or biocompatible collagen. and a transplantation site: and e~cposinp the purified and/or biocompatible collagen to the transplantation site.
DESCRIPTION OF THE FIGURES
Figure 1 is a flow chart of one embodiment of the Twice Treatment ProcessT'"
(TTPTM).
Figure ? is a schematic showing; phosphorylation of serine to phosphoserine.
Figure 3 is a bar chart showing the wound healing properties of various collagen preparations.
Figure ~4 is a graph showing; the wound healing properties and blood clotting times of various preparations.

I ~ The present invention relates to methods for preparing collagen from tissues of humans and other animals. In particular. the present invention provides methods for the preparation of collagen suitable for biomedical, veterinary, and other applications.
The present invention was developed in order to address the problems presented by commonly used collagen preparations. The present invention is predicated in part on the discovery that collagen may be prepared in a manner in which all non-collagenous materials are removed. while retaining the native molecular quaternary structure and other characteristic features of collagen (e.~,~.. length. diameter. and periodicity of collagen type I fibrils. as described above). The methods of the present invention facilitate the enzymatic removal o1' all extraneous materials while preserving the native collagen molecules in their orig=inal fiber ?5 configuration. The processes of the present invention may be used to prepare highly purified collagen from various animal sources (including humans). as most if not all.
conjugated proteolipids and phospholipid contaminating the source collagen are removed through use of a specific mixture of organic solvents. In various embodiments. the prepared collagen of the present invention has better wound healing and hemostatic properties than collagen preparations previously developed.
Unlike previously reported enzymatic methods in which papain is used (c~.~..
U.S.
Patent No. 3,539.530 and 5.316,942) for collagen preparation. the methods of the present invention utilize a two-step enzyme treatment process.

In one embodiment. the two-step treatment process ("Twice Treatment ProcessTM"
or "TTPT"'") of the present invention renders collagen polymers non-inflammatory through the processes in which papain or other proteolytic enzymes are used in conjunction with oxidative and reducing agents. The "twice-treatment" refers to the use of proteolytic enzyme in two steps: a first proteolytic treatment is conducted. followed by treatment with a reducing agent, which is then followed by a second proteolytic treatment. In particularly preferred embodiments. this process is followed by removal of proteolipids and phospholipids using a solution of chloroform and methanol, In additional embodiments. the collagen is bioaetivated by varied degrees of controlled phosphorylation.
I U The use of papain was contemplated as providing a safe means for treating collagen intended for human use, as it is traditionally used in the food industry and in association with wound cleansers. In comparison with pepsin. the enzyme most commonly used to prepare collagen for biomedical applications. better results in terms of reduced immunogenicity was obtained with papain. Papain. an enzyme extracted from papaya, is known to break the 15 disulfide honds of cysteine. As many immunogenic molecules contain cystine disulfide bonds. papain may be used to degrade these molecules and render them non-immunogenic.
F'or example. papain is capable of di~~esting numerous naturally occurring proteins and peptides. as well as benzoyl amino acid esters (See e.~~., Smith and Kimmel, Enzvmes~, The, vol. 4. 2d ed., Academic Press, NY. page 138 [1960]). In addition, papain has been reported ?() to have a Ivtic ettect of elastin, one of the contaminants that is difficult to remove from purified collagen (,See c,~.g.. Coulson, Bioehim. Biophys. Acta 237:378 [ 1971 ~; and Smith e~
cr., Nature !98:1311 [1963]).
Initial experiments involvin~~ a one-step papain treatment to remove immunogenic sites trom collagen were largely unsuccessful in altering the in aivu performance of purified ?5 collagen (.Sce. example 3. below). These observations led to the development of the methods of the present invention. which result in the breaking and loosening of the natural crosslinks of collagen fibers (e. s,~., aldol condensation). In this manner, the papain used in the second treatment (i.e., papain is used in two treatment steps) is provided access to most, if not all of the collagen molecules' surfaces. and facilitates the release of trapped immunogenic sites tr0117 ,0 the collaLen preparation. These developments resulted in one embodiment of the present invention. in which papain is used at two specific stages of the process (i.c~., before and after the treatment the colla~~en with a reducing and/or a unfolding agent). These methods therefore. provide means to produce highly purified collagen that is non-immunogenic.

In a preferred embodiment. a delipidation process is contemplated. in which solvents are used to remove proteolipids and phospholipids. Early attempts to remove these compounds ti-om collagen using single organic solvents) such as ether.
acetone, ethanol.
isopropyl alcohol. etc. were not effective in removing all the proteolipids or conjugated lipids like proteolipids. (,See, Example 4). Therefore. a unique technique was developed lIl \ulllch a s: l v/v mixture of chloroform : methanol was used to remove all the proteolipids and hhospholipids.
In a preferred embodiment, purified collagen may be chemically-modified by covalently binding phosphates to hydroxyl groups of hydroxylated amino acids.
as shown in 1 () Example 6. Although it is not necessary to understand the present invention, this reaction likely involves covalent bonding of phosphate to hydroxyl group of serine.
tyrosine and/or threonine. hvdroxylysine and hydroxyproline. The reaction is controlled. in order to limit the ~le~~ree of reaction. At the completion of this step, any of the potentially remainin~~ reactive ~~roups present in the collagen may be fully converted to phosphoryl.
hydroxyl, or sulfonyl I S ~~roups by exposure to trimetaphosphate or other active agents. The end product with different degrees of chemical modification or without the same that is either soluble or insoluble in a physiological buffer and is suitable for numerous applications.
In this manner, the purified product can be customized to particular uses. For example.
bioactive responses may he favored by reacting the collagen with a linear or cyclic polyphosphates at alkaline pH.
?0 The final collagen product may be used in solution or as a solid. and has been shown to be useful for a variety of purposes. including but not limited to, biological implants. grafts.
transplants, and drug delivery. It is, moreover, contemplated that it is useful as a surgical adjunct during transplant surgery and to prevent post-operative graft dislocation, as a hemostatic agent, to augment soft tissues, and as a support for in vitro cell growth (i.c~., in cell ?J cultures).
When used as a hemostatic scent, the collagen prepared accordin~~ to the methods of the present invention is particularly applicable to the control of bleedings from surfaces.
especially large surfaces. For example. the collagen of the present invention may be used as a hemostatic absent on cut or severed bone, organs (e.g., spleen. liver or kidney which has been 30 cut surgically or traumatically), the central nervous system with its predominant collection of small blond vessels, prosthetic surgery, oozing surfaces resulting from the surgical removal of necrotic tissue. cosmetic surgery. and any surfaces with oozing of blood tcom one or more small sources (e.~l. facial cuts). This hemostatic collagen preparation may be applied in a variety of t01'IllS ( e.,~l.. as a powder applied directly to the surface: as a styptic in pencil form:
as a gel, a sponge or in fabric form). The amount of hemostatic collagen preparation utilized will vary with the extent of the bleeding, the surface area to be treated, and severity of the blood flow: the only requirement being that the achievement of the desired control.
Definitions To facilitate understanding_ of the invention, a number of terms are defined below.
The: terms "specimen"or "sample" are used interchangeably in the present specitlcation and claims and are used in their broadest sense. On the one hand they are meant to include a clinical specimen (i.e.. sample) or culture (e.g., microbiological cultures).
On the other hand.
it is meant to include both biological and environmental samples. Biological samples tnay be animal, including human. fluid or tissue, food products and ingredients.
However, these examples are not to be construed as limiting the sample types applicable to the present invention.
1 ~ The "non-human animals" of the invention comprise any animal other than humans.
Such non-human animals include vertebrates such as rodents. non-human primates, ovines, bovines. ruminants. lagomorphs, porcines, caprines, equines. canines, felines, ayes, etc.
Although it !S llUt intended that the present invention be limited to any particular animal.
preferred non-human animals are selected from livestock, such as bovines and ovines.
As used herein. the term "procollagen" is used in reference to the precursor protein that is cleaved in the extracellular matrix to form collagen. In particular, procoilagen is used in reference to "pro-a chains" that are precursors of the collagen a chains.
1'ro-oc chains are characterized as comprising "pro-peptides" that are important in the formation of the triple-helix formation of procollagen.
As used herein. the term "collagen" is used in reference to the extracellular family of fibrous proteins that are characterized by their stiff, triple-stranded helical structure. Three collagen polypeptide chains ("a. -chains") are wound around each other to form this helical molecule. The term is also intended to encompass the various types of collagen, although the preferred form is type I collagen.
As used herein. the term "collagen sample" refers to any source of collagen.
including.:, but not limited to hide. skin, tendons, blood vessels, intestine. liver.
spleen. heart valve. bone.
etc. It 1S llUt intended that the source of collagen sample be limited to any particular tissue source or type. The only requirement is that the collagen sample contain the type of collagen of interest to be purified (c.,cl., type 1 collagen). However, it is not intended that the present invention be limited to any particular type of purified collagen.
As used herein. the term "collagen fibrils" is used in reference to the long, thin polymers of collagen that are grouped into bundles referred to as "collagen fibers." The "collagen matrix" refers to an extracellular matrix of collagen, with the characteristic periodic cross-striations that may be visible throubh electron microscopy.
Characteristically. the cullas~en matrix is formed when normal collagen molecules are assembled in a quarter stagger array in a three-dimensional structure appearing by electron microscopy as a 67 nanometer strtanon.
As used herein. the term "compressed collagen" refers to a collagen sample that has been compressed due to the application of pressure. In preferred embodiments, the collagen to be compressed is wet. TTPTM-treated, and delipidated collagen. It is intended that this compression be accomplished by any means (i.e.. hand presses or machine presses). It is intended that this compression encompass a range of pressures, ranging trom x,000 to ~O,OOU
1 s pounds per square inch (psi) of sample. In preferred embodiments, the pressure applied is approximately 9.000. while in alternative preferred embodiments. the applied pressure is approximately 30.0U0. In particularly preferred embodiments, the compressed collagen is in the form of a cake.
As used herein, the term "dehydrated collagen" refers to a collagen sample that has been dehydrated using any method commonly known in the art. In preferred embodiments, dehydrated collal;en is produced by lyophilization or desiccation.
.As used herein. the term "drying" refers to any method for the removal of water from a sample. It is intended that the term encompass methods including. but not limited to. air-drying and heating.
?> As used herein, the term "dispersing" refers to the mechanical separation of a sample.
For example, it is intended that the term encompass the separation of the components contained within a cake of compressed collagen. In this embodiment, the components of the compressed cake are dispersed into fibers. In preferred embodiments, the collagen is agitated and suspended within a liquid. These fibers comprise collagen fibers as described above (i.e., 3() bundles of collagen fibrils). Thus. it is intended that native, as well as dried collagen fibers be encompassed by this definition.
As used herein. the term "hemostatic" refers to an agent that stops or slows the flow of blood.

As used herein. the term "proteolytic agent" refers to any enzyme ( alone or in a mixture of enzymes) that is capable of hydrolyzing or cleaving proteins. It is intended that the term encompass any enzyme with proteolytic properties. including. but not limited to the cvsteine class ol~ enzymes which reacts with cysteine moieties in their substrates. This class of enzymes includes. but is not limited to glyceraldehyde-phosphate dehvdrogenase. and papam.
in particular. it is contemplated that the term refer to enzymes that are capable of degradin~~ proteins associated with collagen, while leaving collagen type I in its native conti~~uration. 1-Iowever, it is contemplated that in some embodiments. the proteolytic agent breaks the cross-links associated with collagen fibers in collagen. In this manner. the cross-links between the strands within the fibers are loosened, such that the fibers retain their basic shape, but sufficient space is allowed between the fibers that molecules that are normally trapped within the collagen tlber structure may be removed.
As used herein. the term "proteolyzed collagen" refers to collagen that has been treated with at least one proteolytic enzyme.
As used herein. the term "purified" refers to the removal of contaminants from a sample. For example. it is intended to encompass proteolyzed collagen. It is not intended that the term be limited to any particular level of purity. I-lowever, in preferred embodiments, the purified sample is treated with proteolytic enzymes. In particularly preferred embodiments. the purified sample has undergone subsequent treatments.
including but not limited to. delipidation and/or phosphorylation.
As used herein, the term "reducing agent" refers to any compound or mixture of compounds that is capable of reducing another compound. In this process the proportion of hydrogen is increased while the proportion of oxygen is decreased and the number of multiple ?5 bonds is decreased. In particular. it is intended that the term encompass any agent that is capable of unfolding proteins from their native configurations by breaking disulfide bonds.
Such absents include, but are not limited to compounds such as sodium sulfide, dithiothreitol, 5lutathionine, and sodium borohydride. The term "reduced collagen" refers to collagen that has been exposed to or treated with at least one reducing agent.
s() As used herein. the term "phosphorylation agent" refers to any compound or mixture of compounds that is capable of phosphorylating another compound. Such compounds include. but are not limited to sodium trimetaphosphate. sodium hexametaphosphate. sodium ultraphosphate, sodium tetrametaphosphate. phosphoric anhydride, and phosphoryl trichloride.

As used herein. "phosphorylated collagen" refers to coflaeen that has been treated so as to increase the number of phosphorylated amino aids in the collagen molecules.
It is intended that the term encompass a range of phosphorylation, from a minimal degree of phasphorylation (i.e.. only one amino acid has been phosphorylated) to a maximal degree of phosphorylation (i.e., all of the amino acids available and suitable for phosphorylation have been phosphorylated), or any degree of phosphorylation within this range.
As used herein. the term "ctelipidation agent" refers to any compound or mixture of compounds that is capable of removing lipid moieties (i.e., delipidatin~;). In particular it is intended that the term encompass substances that are capable of removin';
molecules 1 U including. but not limited to, phospholipids, neutral lipids, proteolipids. and glycolipids from collagen. Such substances include organic solvents such as chloroform and methanol. The term "delipidated collagen" refers to collagen that has been exposed to or treated with at least c~nc; delipidation agent. In particularly preferred embodiments. the delipidation agent is chloroform and methanol in a 3:1 mixture.
I 5 As used herein, "alkali" refers to a hydroxide of one of the alkali metals (e. s,~., lithium, sodium, potassium, rubidium, cesium. and francium). It also encompasses substances which provide an alkaline solution (pH > 7) in water (c~.~,~., CaO, Ca(OH),, and Na,CO~.
As used herein, "modified collagen" refers to collagen that has been chemically modified by such means as phosphorylation, fluorination, halogenation (c~.~~., chlorination), 30 suifonation, and/or hydroxylation.
As used herein. the term "filter-sterilized" sample refers to a sample that has been applied to a filter to remove unwanted contaminants such as bacteria. from the sample.
As used herein. "de-epithelialization" refers to the process of removing epithelial tissues and/or cells from a sample. The term "epithelial" pertains to epithelium, the cellular ?5 covering of internal and external body surfaces.
As used herein. "biocompatibie collagen" refers to collal;en that is suitable for biomedical or veterinary applications. including but not limited to grafts.
and implants.
As used herein. "biomolecule" refers to any molecule that has biomedical or veterinary applications. For example, it is intended to encompass various compounds and substances, s0 including collagen. synthetic polymers. or other materials useful for implantation, transplantation, and/or grafting.
The term "graft" refers to anv tissue or organ for implantation or transplantation. as well as the process of implantation or transplantation of such tissue. For example, a skin - l~ -~~ratt is a piece of skin or other tissue or material that is transplanted in order to replace a lost porton of the shin. It is intended that the teen encompass materials such as purified collagen ur other suitable biocompatible substances useful in the replacement of lost or damaged tissues or organs. In particular, the term "graft" is used in reference to the replacement of skin or other tissues or organs exposed to the environment. For example. the term may be used in reference t0 SIICh tissues as skin. cutaneous tissues. mucous membranes, conjunctiva) membranes. etc. However, it is not intended that the term be limited to anv particular type of tissue.
The term "transplant" refers to tissue used in grafting. implanting. or transplanting, as well as the transfer of tissues from one part of the body to another. or the transfer of tissues from one individual to another, or the introduction of biocompatible materials into or onto the body. The term "transplantation" refers to the grafting of tissues from one part of the body to another part, or to another individual. However. it is also intended that the tissue or organ used for transplantation or grafting be comprised of materials such as collagen purified f s according.: to the methods of the present invention. Thus. it is not intended that the braft or transplant be limited to naturally-occurring tissues.
As used herein. the term "implantation" refers to the insertion of an organ or tissue in a new site in the body. as well as the insertion of any biocompatible, biological, living, inert, or radioactive material into the body. The term "implant" refers to the process of inserting or grafting of tissue, inert. living, radioactive, or biocompatibte material into intact tissues or a body cavity, as well as the material to be so inserted or grafted.
As used herein, the term "flap" refers to a mass of tissue for grattin~" which usually includes skin, that is only partially removed from one part of the body, so that it retains its own blood supply during transfer to another site of the body or to another individual. It is also intended that the term encompass masses of tissue for grafting or implantation that have been cultivated in vitro prior to their grafting or implantation.
EXPERIMENTAL
The tollowing examples are provided in order to demonstrate and further illustrate i0 certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents): M (Molar); p.M (micromolar); N (Normal); mol (moles); mmol (millimoles);

lrmol (micromoles): nmol (nanomoles); g (grams): mg (milligrams): Erg (micrograms); ng (nanograms): 1 or L (liters); ml (milliliters); ~1 (microliters): cm (centimeters); mm (millimeters): ym (micrometers); nm (nanometers); A (angstrom); xg or x ~
(times gravity);
w/w (weight to weight): w/v (weight to volume);°C' (degrees Centigrade): rpm (rotations per minute): normal saline (0.9% NaCI solution, pH 6.:.~ -~ 0.3); Harlan SpraLUe Dawley (Harlan Sprague Dawley. Inc.. Madison. WI); Fisher (Fishe:r Scientific. Pittsburgh, PA); SiLma (Sigma Chemical Co.. St. Louis. MO.): Aldrich (Aldrich Chemical Company. Inc..
Milwaukee. WI):
Spectrum (Spectrum Chemicals, Inc.. Gardena, CA); American (American Dade, Division of American Hospital Supply Co.. Dade, FL); PDL (Protein Design Laboratories.
Mountain View. CA): Nitabell Rabittry (Nitabell Rabittry, Hayward, CA); Axygen (Axygen.
Inc..
I-Iayward. CA): Arizona Health Sciences (Arizona Health Sciences Center.
Tucson. AZ);
Collaborative Research (Collaborative Research. Inc., Bedford, MA); Medchem Products (Medchem Products. Woburn, MA): Collatek (Collatek, Inc.. Plainsboro. NJ):
Boehringer Mannheim (Boehringer Mannheim. Indianapolis. IN); J & J Medical (J R; J
Medical, Inc., Arlington, TX); Axygen (Axygen Inc.. Hayward, C_'A); Papain Products (Papain Products, Tamiinadu, India); Beckman (Beckman Instruments, Inc.. Fullerton, CA); and Brinkmann ( Brinkmann I nstruments. Inc., Westbury, NY).
Unless otherwise indicated, all chemicals were obtained from commercial suppliers such as Sigma, Fisher. or Spectrum. All of the solvents used in these Examples were obtained from Spectrum Chemicals, and were spectrophotometric or reagent grade. The papain used in these Examples was obtained from Sigma or Papain Products.

Isolation And Purification Of Collagen With Papain ?5 In this Example, collagen was purified from dehaired bovine hide. using a one-step enzyme treatment process.
First. 100 g of clean. dehaired bovine hide obtained from a local slaughterhouse was chopped or sliced into pieces of approximately 1 cm thickness and approximately 10 cm long.
The cut slices were washed in three liters of a wash buffer containing double distilled water containing 30 : NaCI. , ~ ZnCI. 4.~ g methyl paraben. 0.9 g propyl paraben. In some experiments. the wash buffer contained 30.0 g NaCI. 3 g ZnCI. 4.~ g methyl paraben, and 1.5 ~~ trichlorophenate or , g hypochlorite. No differences were observed in the purified collagen preparations using these different wash buffers. ~YashinL was conducted at 5-1 ~°C for 6-8 hours under constant agitation using a Rotator (Model #4140. American). The wash procedure was repeated twice, with the fluid being decanted from the solids after each wash.
The repeated wash procedure removed most of the albumins and globulins that usually > constitute approximately I-5% of the dry weight of the sample.
Prior to application of tlje enzyme treatment. de-epithelialization of the sample was conducted. In this process. 1 liter of cold de-epithelialization butter containing cold deionized water containing, 24 g NaOH and ~6 g calcium oxide was added to the previously washed sample. and centrifuged at 5.000 xg for 15 minutes to obtain solid chunks of material. The sample was agitated on a rotator at 80-100 rpm, for 48 hours at 15°C.
The de-epithelialization solution was replaced every l6 hours. At the end of 48 hours, the chunks were soaked in 3 liters of deionized water for 2 hours, with agitation at 80-100 rpm. and the solids were recovered by centrifu~~ation at 5,000 xs~ for 1 s min. One hundred grams of boric acid solubilized in 3 liters of deionized water pre-chilled to 4°C. was added and the sample 1 ~ was agitated for 16 hours at 4°C. The pH was measured and adjusted to approximately 8.3.
The sample was washed twice with 3 liters of deionized water for 2 hours each wash, and the solids were recovered by centrifugation as described above. Although this method was used in this Cxample, various methods for achieving de-epithelization of samples are available. and known to those in the art. It is contemplated that any of these standard methods available to those in the are will be successful in achieving the desired de-epithelization.
After washing, the sample was treated with papain in a one-step enzymatic treatment.
In this process, the sample was further washed in 3 liters of pyrogen-tree deionized water for 2 hours at room temperature. The sample was centrifuged for 1 ~ minutes at ~,OOU xg, and decanted. One liter of normal saline solution was then added to the sample.
Papain (Sigma ?5 or Papain Products) was added at the rate of 1-2 mg per ml of the total volume (i.c~.. 1 liter), with continual stirring. Following addition of the papain. the solution was incubated under agitation using a rotator at 80-100 rpm. at 37°C for approximately 16 hours.
Following this tirst enzyme treatment, the excess water was drained and the solids were washed three times with 2 liters of deionized water. One liter of 0.2 M
sodium borohvdride was then added to the washed solids. The solids were incubated for 3 hours under agitation with a rotator at 80-100 rpm. The excess water was drained.
and the solids were washed three times with 2 liters of deionized water.

WO 98/30225 PCTlUS98/00371 Robust Treatment In addition to the normal enzyme treatment followed by reducing treatment. as described above, a "Robust Treatment" was also tested. In this treatment. a multifold increase in time of incubation and enzyme concentration were used. as compared to the "normal"
treatment. Although the rest of the steps were the same as described far the normal treatment ( i. e., the cailagen used in the "Robust Treatment" was prepared using the same bovine hide.
ruttin~~ and \~'~tshln ~._' steps). in the Robust Treatment. 4 mg of papain was added per ml of solution and ?4 hours of incubation were used in the enzymatic treatment step, rather than the 1 mg of papain per ml of solution and 16 hours of incubation.
The amount of remaining contaminants and collagen in this preparation was determined and compared with the results obtained using the methods described in Example 2.

Isolation And Purification Of Type 1 Collagen This Example describes the methods and aspects of the development of the "Twice-Treatment ProcessTM" (TTPTM) of one embodiment of the present invention.
In this Example, the "Twice-Treatment ProcessTM" (TTPTM) is described. In this Example. type 1 collagen was purified from dehaired bovine hide. First. 100 g of clean, dehaired bovine hide abtained from a local slaughterhouse was chopped or sliced into pieces of approximately 1 cm thickness and approximately i0 cm long. The cut slices were washed in three liters of a wash buffer containing double distilled water containing 30 g NaCI, ; g ZnCI. 4.5 ~~ methyl paraben. 0.9 g propyl paraben. In some experiments. the wash buffer contained 30.U g NaCI, 3 g ZnCI, 4.5 g methyl paraben, and 1.5 g trichiorophenate or 3 g ?5 hypochlorite. No differences were observed in the purified collagen preparations using these different wash buffers. Washing was conducted at 5-15°C for 6-8 hours under constant agitation using a rotator (Model #4140, American) at 80-100 rpm. The wash procedure was repeated twice. with centrifugation at 5,000 x g for 15 minutes. and decanting of the supernatant each time. The repeated wash procedure removed most of the albumins and ;0 !~lobulins that usually constitute approximately 1-5% of the dry weight of the sample.
Prior to application of the enzyme treatment, de-epithelialization of the sample was conducted. In this process. I liter of cold de-epithelialization buffer containing cold deionized water containing ?4 ~~ NaOH and ~6 g calcium oxide was added to the previously washed sample. and centrifuged at x.000 xg for 15 minutes to obtain solid chunks of material. The sample was agitated on a rotator at 80-100 rpm, for 48 hours at 15°C.
The deepithelialization solution was replaced every 16 hours. At the end of 48 hours. the chunks were soaked in 3 liters of deionized water for 2 hours. with agitation at 80-100 rpm, and the solids were recovered by centrifugation at x.000 xg for 1 S min. One hundred grams of boric acid solubilized in 3 liters of deionized water pre-chilled to 4°C, was added and the sample was agitated for I6 hours at 4°C. The pH was measured and adjusted to approximately 8.3. The sample was washed twice with 3 liters of deionized water for ? hours each wash, and the solids were recovered by centrifugation as described above. Although this method was used in this Example. various methods for achieving de-epithelization of samples are available. and known to those in the art. It is contemplated that any of these standard methods available to those in the are will be successful in achieving the desired de-epithelization.
After washing. the sample was treated with the TTPT"'. In this process, the sample was further washed In 3 liters of pyrogen-free deionized water for 2 hours at room I S temperature. and the water was decanted. One liter of normal saline solution was then added to the sample. Papain was added at the rate of 1-2 mg per nil of the total volume (i.c~., 1 liter), with continual stirring. Following addition of the papain, the solution was incubated under agitation on a rotator at 80-100 rpm, at 37°C for approximately 16 hours.
Following this first enzyme treatment, the excess water was drained and the solids were washed three times with 2 liters of deionized water. One liter of 0.2 M
sodium borohvdride was then added to the washed solids. The solids were incubated for 3 hours under agitation with a rotator at 80-100 rpm. The excess water was drained, and the solids were washed three times with 2 liters of deionized water. The second enzyme treatment was then conducted on the solids, using the same procedure described above for the first enzyme treatment. A strong reducing agent was found to be essential. Although it is not essential to understanding or using the present invention, it may be that this strong reducing; agent stabilizes any reversibly reactive group that might remain following the first enzyme treatment. This step also reduced the amount of aldehyde potentially involved in aldol condensation reactions (l. e., reactions between two aldehyde groups) or Schiff base reactions s0 ( l. c'.. reactions between an aldehyde group and an amino ~_roup (Sc~e c.g.. Nimni e1 crl.. "Bioprosthesis derived from crosslinked and chemically modified collagenous tissues." in C.'nllcr,~len, vol. 3. M.E. Nimni et crl., CRC Press, Inc.. Boca Raton. FL
[1988], p. ~). It may also reduce the interchain disulfide bonds in the helical region of the same molecule, present only in tvpe III collagen (.Sec~ e,r., D. Amiel et crl., "Biochemistry o1' tendon and ligament." in ( 'ullu~,~en, vol. 3, p. 229. .~~trpra).
Following the second enzyme treatment. the excess water was drained, and the solids were washed three times with 2 liters of deionized water. Although it is not necessary to an understanding of the present 111Velltloll, it may be that the first enzyme treatment liberates the colla~.:en in the preparation to an extent that is susceptible for further attack by a reducing agent (e.,sr.. SUdlllill SUIt'lde, thiothreitol. dithiothreitol, glutathione or sodium borohydride), with or without alkali compounds to swell the collagen. Following chemical treatment, a second treatment with papain was conducted, in order to thoroughly eliminate all of the residual non-collagenous contaminants (c~.~., interstitial elastin).
Collagen prepared accordinb to the two-step enzymatic treatment, as well as collagen prepared with the one-step enzymatic trearinent described in Example 1 was tested to determine the degree of removal of non-collagenous fibrous proteins (e.,~~., elastin) as described in Example 3.

Insoluble lYon-Collagenous Protein (Elastin) Content In this Example. various samples of collagen preparations were tested for the amount of non-collagenous insoluble protein present, using the methods described by Soskel et u1.
N.T. Soskel cu crl.. Meth. Enzymol., 144:199 (195'.'.]). The samples tested in this Example were treated as described below.
Briefly, approximately 2~ g of wet test sample were delipidated using 2:1 chloroform:methanoi (v/v). The samples were frozen in liquid nitrogen and crushed into pieces. The pieces were then placed in 100% isopropyl alcohol to prevent bacterial growth.
The pieces were then centrifuged at 5,000 xg for 15 min. the supernatants were decanted, and the pellets were air dried at 60°C. The dried samples were then brought to room temperature in a desiccator and the approximately 10 g of each sample were placed in 200 ml polypropylene tubes. One hundred Illl of 0.1 N NaOH at 4°C. was added to each tube and the contents were mixed. The tubes were then placed into a boiling water bath for 4~
minutes. As elastin degradation begins to occur after 60 minutes. it was determined to be important to remove the tubes from the boiling water bath after 4~ minutes.
The samples were then washed with cold 0.1 N NaOH, and then washed with cold double distilled water.

'The samples were dried over P,O; or lyophilized, in order to avoid the inclusion of moisture in the samples that would result in a higher weight measurement. The samples were kept in a desiccator until use. The samples were brought to a constant weight (i.c~., the samples were repeatedly weighed until the weight no longer varied) for ~~ravimetric quantification.
s In Tabie 1. the results are shown as percent dry weight from 10 '~ of dry starting sample. (n this Table. results are shown from samples prepared using the "normal" and "robust" papain enzyme treatments (i.e., the robust treatment described in Example 1 ). In this Table. "Enzyme Only" refers to a sample that processed according to the method described in Example 1 (i.e.. it was processed without any reducing agent--the first enzyme treatment was conducted and the treatment process was halted).
In this experiment. samples prepared according to the TTP method described above were tested. as well as samples prepared according to the "Robust Treatment"
described in Example 1. In addition. two other samples were tested using both the normal treatment and robust treatments. The "Enzyme-Reducing Agent" sample refers to a sample that was tested IS following one enzyme treatment and one reduction step (i.e.. the first enzyme treatment of Example ?. followed by the reducing step of Example 2).
The sample indicated as "Reducing Agent~Enzyme" refers to sample that was tested following one reduction step followed by one enzyme treatment. This "Reducing Agent~Enzyme" sample was prepared from the same source of collagen. cut, and washed as described in Examples 1 and 2. The test sample was first reduced using the reduction method described in Example ?. but without the first enzyme treatment. Following this reduction step, the 111etI7odS described for the first enzyme treatment were conducted on the reduced sample.
The sample indicated as "TTPTM-Treated" was a sample prepared according to the methods of Example ?. As no insoluble non-collagenous protein was detected using the normal methods of "TTPTM-treatment, the robust version of this process was not conducted.
'the characteristics of this TTPT"'-treated sample are discussed in more detail in Example ~
below.
The untreated control contained 1.24 ~ 0.190 percent dry weight of non-collagenous insoluble protein. while the sample prepared by the TTP process, had no detectable dry weight non-collagenous insoluble protein. These results indicate that there was a significantly ~~reater degree of removal of extraneous non-collagenous proteins in this experiment. as compared to collay'en prepared with only one enzyme treaunent step, as described in Example Table I. Non-Colla~enous Insoluble Protein Concentration Sample Normal Treatment Robust Treatment J Enzyme Only 0.31 0.020 0.27 O.OIG

f-_nzvme->R~ducin~~ 0.30 0.027 0.25 0.014 A~_ent Reducin~~ A';ent-~Enzytne0.19 0.013 0.31 0.029 TTPT"'-Treatment Not Detected Not Conducted Delipidation Of TTPT"'-Treated Collagen In this Example, collagen treated with TTPT"', as described in Example 2 was delipidatcd. in under to remove additional potentially immunogenic sites from the TTPT"'_ I s treated colla~:en.
Fractional Salt Precipitation First, the TTPTM-treated type 1 collagen was separated by fractional NaCI salt precipitation as described by Hill and Harper (R.J. Hill and E. Harper, Anal.
Biochem., 141 :8J-9J ( 1984]). Briefly, the enzyme-treated collagen was dissolved in 0.5 M NaCI in Tris (hydroxymethyl amino methane buffer), at pH 7.4. The NaCI concentration was then increased to 1.7 M. The solution was mixed and centrifuged at 10,000 x~~ for 1 hour at 0-4°C. The dewatered precipitate was mixed and solubilized in 0.5 M
acetic acid, and the pH
was adjusted to 7Ø Sodium chloride was added to a final concentration of 2.5 M, and the precipitate was collected by another centrifugation step as above.
The precipitated collagen was then dialyzed against deionized water and lyophilized prior to delipidation for quantitative analysis of the lipid content in the collagen sample.
In later experiments. it was found that this fractional salt precipitation was often not necessary. as it did not atfiect the purification level of the TTPTM-treated type l collagen protocol. I-Iowever. it is intended that this step may be conducted prior to delipidation.

Delipidation The collagen preparation was then dilipidated using the method of Folch et up.. (Folch m up.. J. Biol. Chem.. ??6:497-509 [1957]), with modifications. This step involved solvent extraction of total lipids. including gangliosides and phospho-inosities.
Briefly, the collagen preparation was stirred in a mixture of chloroform and methanol (2:1, v/v).
The volume of the chloroform: methanol was 20 times that of the collagen sample (i.c-.. ?0 parts of the chlorol-orm:m~thanol mixture were added to 1 part of collagen sample). The sample was atirred well during this addition, and vigorously shaken for one hour at 40-45°C.
The homogenate was transferred to a vial, and 0.? volumes of 0.88% I<Cl were added.
'fhe two phases of the mixture were allowed to separate in a separating tunnel. The lower phase containing major lipids was removed, and the upper phase, which contained the collagen was washed twice with a mixture of 3:48:47 (v/v) chloroform:methanol:water. The collagen preparation was then washed with a mixture of x:80:15 (v/v) of the same components (i.e.. chloroform:methanol:water), and dewatered using a pressure of 9.000 ~1000 psi, using a hand press. This squeezed the water from the collagen preparation to produce a dewatered mass. However, it is contemplated that pressures ranging from approximately 8000 psi to 30.000 psi are suitable for use in this step. Application of pressure at 8000 psi removes all of the extraneous water from outside of the molecules. while application of approximately 30.000 psi removes even the intramolecular water. However. it is not intended that the present invention be limited to these specific pressures.
The compressed cake was then dispersed, using a coffee grinder. and air dried in a ventilated drying oven at 40-45 °C. The purity of the delipidated sample was then tested for phospholipids, neutral lipids and l;lycolipids as described below.
Thin layer chromatography (TLC) was used as described by Skipski ce crl..
(Skipski c~t crl.. Biochem. .I.. 90:374-378 [1964]) to detect phospholipids in the samples.
Briefly. 10 g of delipidated sample was tested using a silica gel TLC method. The TLC plate was prepared by first preparing a slurry of 45 g of silica gel H (Spectrum) in 65 ml deionized water. The slurry was poured onto a clean glass plate (20 cm x 20 cm), to a thickness of 0.5 mm, air dried and activated (i.e.. cured) for 60 minutes, at 60°C, cooled to room temperature, and used immediately. A known quantity of extracted lipid (200 y1) was applied as a streak to the dried silica gel, approximately 2 cm from the bottom of the plate. The sample was allowed to air dry and then was run in an irrigating medium of chloroform:methanol:aqueous ammonia (28%). at a ratio of 65:2:5 (v/v) in a glass chamber. When the solvent front rc;ached near the top of the plate. the plates were removed ti-om the glass chamber and air dried. The plates were then developed with iodine vapor. Yellowish-brown spots appeared at locations where phospholipids were present. Quantification of the phospholipids present was conducted using the methods of Fislce and Subbarow, as described in Example 7, below. The results are shown in Table 2, below.
The presence of neutral lipids and glycolipids in the TTPTM-treated collagen was determined using a silicic acid column chromatographic method. as described by Rouser cu ul.
(G. Rouser eml., Lipids 2:37-40 [1967]). Briefly, a column (10 mm x ?0 mm) containing silicic acid (Spectrum) was prepared and 10 g of the delipidated collagen was applied in i0 sequential portions of ? grams each, to the column. Thus, the total 10 4~
sample was divided into tive runs on the column; all of the eluted samples were pooled. The sample was eluted with 1O0 ml of chloroform:acetone (1:1, v/v), followed by a series of acetone and acetone:water solutions. The eluted neutral and glycolipids were collected from the column.
air dried. and estimated gravimetrically. The results are shown in Table 2.
15 For purposes of comparison. multiple samples of the lyophilized collagen samples prepared prior to the delipidation step described above, were subjected to lipid extraction using different organic solvents. Fifteen grams of dry sample were placed in multiple containers. Then, I 00 ml of each of solvent was added to each container ( 1.
c~., one solvent was added to each sample). The solvents used in this experiment were ether, hexane, 20 methanol. ethanol, isopropyl alcohol, benzene, chloroform, acetone, and chloroform:methanol (2:1 ). The containers were incubated at approximately 40°C, with agitation using an orbital rotator at 100 rpm, for 6 hours. The solvents were then separated from the collagen samples by centrifugation and evaporated. Ten grams of each delipidated and dried samples treated with the various solvents were used in the lipid estimations as described above. The results ?5 are shown in Table 2. below. In this table, the percentages are expressed as the percent of total drv sample weight of collagen.

Table 2. Evaluation Of Delipidation Methods Delipidation Method /. Phospholipids /. Neutral Lipids & Glycolipids Chloroform:Methanol <0.0001 ~r0.0001 (2:1) Ether 0.0210 0.0037 Hexane 0.0260 0.003 I

Methanol 0.0190 0.0039 I~tltanol 0.0200 0.003() Isopropyl Alcohol 0.0190 (1.0030 t3enzene 0.0230 0.0029 Chloroform 0.0240 0.0030 Acetone 0.0 t 70 0.0028 As indicated in Table 2, no detectable quantities of phospholipids. neutral lipids, nor ~~lycolipids were detected in the chloroform:methanol extracted samples produced as described l ~ above. On the contrary. the same protein samples showed detectable quantities of phospholipids. neutral lipids and/or glycolipids, when the delipidation was done by traditional methods (e.h., using a single solvent system such as ether, hexane, methanol, ethanol.
isopropyl alcohol, benzene. chloroform. or acetone). These phospholipids, neutral lipids and/or glycolipids are detectable in these samples even after prolonged extraction (c~.~J., several days) at room temperature or slightly higher temperatures (c~.~~., 50°C).

Cysteine, Hydroxyproiine And Hexosamine And Elastin (Insoluble Non-Collagenous Protein) Content Of TTPTM-Treated Collagen In this Example, the cysteine, hydroxyproline, hexosamine. and eiastin content of delipidated and TTPTM-treated collagen was determined. The hydroxyproline content is recognized as a measure of the amount of collagen present in a sample.
The delipidated and dehydrated type I collagen sample was reconstituted in 0.1 M
acetic acid, to a concentration of 0.3% (w/v). The purity of the collagen was evaluated by I-fPLC to determine its amino acid content using a Beckman 6300 amino acid analyzer.
according the manufacturer"s instructions. The cysteine content of the sample was determined. as was the cysteine content of a collagen preparation that was "less phosphorvlated." as described in Example G. These results indicated that the mole percentage of both the TTPTM-treated collagen and "less phosphorylated collagen" was 0.09.
In addition. the insoluble non-collagenous protein (i.c~., elastin) content of TTPT'~-treated colla~~en was determined using the methods described in Example 3 for testing the s insoluble non-collagenous protein content of collagen. ns elastin was the only non-collagenous protein potentially remaining in the TTPT"'-treated type I
collagen preparation, it was considered important to determine the elastin content of this preparation, in comparison with collagen treated using other methods (v.g., the phosphorylated colia~~en of Example G).
The results of these experiments are shown in Table 3, below. These results are expressed as a percenta~~e (wt~wt) on a dry weight basis.
The hydroxyproline and hexosamine contents of the delipidated and dehydrated type I
collagen, as well as collagen prepared according to the methods described in U.S. Patent No.
s.374.~39. and two phosphorylated collagen samples ("more phosphorylated" and "less phosphorylated") described in Example G, were tested in the following experiments.
l~
Determination Of Hydroxyproline Concentration The hydroxyproline content of the delipidated and dehydrated type I collagen.
as well as collagen prepared according to the methods described in U.S. Patent No.
5,374.539. and two phosphorylated collagen samples ("more phosphorylated" and "less phosphorylated") described in Example G, were estimated using; the method described by Stegeman (H.
Stegeman. J. Physiol. Chem., 311:41 [1958]). The 0.03 M chloramine T used in this Example was prepared by added 0.845 g chloramine T to 100 ml of a butter comprised of 20 ml water. 30 ml propanol. and 50 ml citrate-acetate buffer, pH G. The citrate-acetate buffer (pH G) was prepared by adding 50 g citric acid (1 HBO}, 12 ml glacial acetic acid, 120 g ?s sodium acetate (3 H,O), and 34 g NaOH to 500 ml water. The volume was adjusted to 1 liter, and the pH was adjusted to G. Perchloric acid (3 M) was prepared in 2~.5 ml 70%
HC10~, and the volume adjusted to 100 ml in water. The hydroxyproline stock was comprised of ~ yg hydroxyproline per ml water. ~f he hydroxyproline standards were prepared by adding ?0 mg hydroxyproiine per 100 ml water and 0.05 ml concentrated HCI.
The hvdroxyproline concentrations were determined as follows. One ml of each sample and i ml of standard solution containing 2-5 p.g hydroxyproline were added to 1 ml chloramine solution in separate tubes, and mixed for 20 minutes at room temperature. One ml perchloric acid was then added to the tubes, and mixed at room temperature for 5 minutes.
7_ C)ne ml of ~'% p-dimethylaminobenzaldehyde in propanol was added to each tube.
and mixed for 18 minutes in a 60°C water bath. The tubes were cooled to 20°C, and optical density was read at ~i0 11111. The results are shown in Table 3. below.
Determination Of Hexosamine Concentration The total hexosamine concentration of the delipidated and dehydrated type I
collagen.
as well as collagen prepared according to the methods described in U.S. Patent No. 5.374,5?9.
and Uvo phosphorylated collagen samples ("more phosphorylated" and "less phosphorylated") described in Example 6, were also estimated, using the method described by Blumenkrantz et crl. (N. Blumenkrantz and G. Absoe-Hansen, Clin. Biochem., 9:269 [1976]).
The trisodium phosphate-potassium tetraborate solution used in this experiment was prepared by mixing 98 ml of 1 N tribasic sodium phosphate and 0.5 N potassium tetraborate.
This trisodium phosphate;-potassium tetraborate solution was then used as the solvent for the acetylacetone mixtures for determination of hexosamine in the samples.
Acetylacetone 1 was prepared as a 3.5% (v/v) solution in sodium phosphate-potassium borate buffer.
Acetylacetone II was prepared as a 3.5% (v/v) solution in 0.5 N potassium tetraborate. The Erlich's reagent used in these Examples was prepared by adding 3.2 g p-dimethylaminobenzaldehyde in 30 ml 12 N HCI. diluted with 210 ml 2-propanol.
Briefly, the delipidated sample was placed into a tube. 6 N HCI was mixed into the sample, and the sample was allowed to hydrolyze for 30 minutes at 120°C
(or 12-14 hours at 100°C). The sample was then evaporated to dryness. Concurrently, standards of glucosamine and galactosamine (2.~ to 40 ug for each) were hydrolyzed and evaporated to dryness using the same methods. The samples and standards were resuspended in double distilled water.
1 n the assay to determine the amount of total hexosamine, 0.8 ml of sample or standard was added to 0.6 ml 3.5% acetylacetone I in separate test tubes. and mixed. The tubes were heated at 100°C for 30 minutes, and cooled. Then, 2 ml Erlich's reagent was added to each tube and mixed. The tubes were incubated for 5 minutes and the optical density at ~3~ nm was determined.
in the assay to differentiate between glucosamine and galactosamine, 0.8 ml of sample s0 or standard was added to 0.6 1111 3.~% acetylacetone II in separate test tubes, and 1111Xed. The tubes were allowed to sit for 2 hours in crushed ice, followed by heatin~~ at ~0°C for 15 minutes. Then. 2 nil Erlich's reagent were added to the tubes and mixed. The tubes were incubated at room temperature for 30 minutes and the optical density at X30 nm was _?g_ determined. The results are shown in Table 3. below. The results are shown as a percentage (wt/wt) on a dry weight basis.
Table 3. Elastin, Hydroxyproiine And Hexosamine Concentration Determinations Sample Elastin (% HYdroxyproiine Hexosamine wt/wt) ('%.

wt/wt) (% wt/wt) ~ Raw, Untreated _ t3ovim ~hISSllt'. 0.95 3.27 1.24 Control TTPT"1-Treated BovineNot Detected 9.83 Not Detected Collagen tJ.S. Patent 0.02 9.45 Not Conducted No. 5.374,539 Collasen 1 () Less Phosphorylated Not Detected 9.76 Not Detected Collagen More Phosphorylated Not Detected 9.75 Not Detected Collagen Chemical Modification Of Filter-Sterilized Collagen Type I
l~
In this Example, 'TTPT"'- treated collagen prepared according to the methods described in Example 5 were further treated to produce "CollagenPROT"'." Thus, as it is used in the present invention, "CollagenPROT"'" refers to purified collagen that has been phosphorylated.
CollagenPROT"' was prepared from TTPTM- treated collagen prepared according to the 20 methods described in Example 5. First, TTPTM- treated collagen was filter sterilized by passing the preparations through a 0.22 p filter attached to a 30 ml syringe.
The filter-sterilized collagen suspensions were then stored until use at 4°C.
These filter-sterilized collagen preparations were chemically modified in order to determine the optimum modifications for production of purified collagen suitable for biomedical applications. The 25 entire procedure was conducted under sterile conditions, using pre-sterilized ingredients and pyrogen-free, sterile water.
First_ l00 ml of a 0.3% solution of collagen in 0.1 M acetic acid was treated with 5 N
NaOH at 4°C. until the pH reached 1 1.5. Then, sodium trimetaphosphate was added to a tinai concentration of 0.03 M. The collagen was allowed to react in a sterile hood kept at 30 room temperature for approximately 3 hours with agitation using an orbital rotator, at 160 rpm. This modified collagen was washed by dialysis against deionized water at 4°C, until no residual ions leached out of the collagen sample. Portions of the modified collagen were dried by lyophilization (i.e., freeze-drying) using methods commonly known to those in the _ 29 .

art. The lyophilized samples were stored in a desiccator for a minimum of five days prior to additional testing.
For some experiments. it was of interest to include purified collagen with a varying ~ie~~rees of phosphorylation. For samples with more phosphorylated collagen.
referred to as "More Phosphorylated Collagen" in the accompanying Examples, approximately 40%
of the modifiable sites available in the collagen were phosphorylated. For samples with lesser cle~;ree of phosphorylation; this preparation is referred to as "Less Phosphorylated Collagen" in the accompanying Examples. To achieve a purified collagen with less phosphorylation. 0.01 v1 sodium trimetaphosphate was used in the phosphorylation step, rather than 0.03 M sodium trimetaphosphate. In addition. the incubation period was 1 hour. rather than the 3 hours used with 0.03 M sodium trimetaphosphate.
It is contemplated that phosphorylation of biomolecules. such as growth factors (e.~..
IGF), interleukins (c.,~,r.. IL 1 ), and other biologically active molecules is beneficial. For phosphorylation of EGF. 1 ml of 0.1 % EGF adjusted to pH 1 1.5, is added to 0.2 1111 sodium 1 s trimetaphosphate. at the same concentrations and conditions (e.g..
agitation and temperature).
as described above. The sample is then dialyzed to remove salts, and the phosphorylated EGF
may be used as appropriate.

Testing For Covalently-Bound Phosphorous In this Example. washed collagen sample was tested for the presence of covalently bound phosphorous. using the procedure described by Fiske and Subbarow (C.I-i.
Fiske and Y.
Subbarow. ,1. Biol. Chem., 66:375-400 [1925]). Briefly, 1 mg of dry modified collagen prepared and lyophilized according to Example 6 was mixed with 1 1111 of 72%
perchioric acid, and then digested at 200°C, for 20 minutes. Deionized water was added to the digested sample to adjust the volume to 4 ml. Then, the following reagents were added in succession, and mixed well. First. U.4 ml of 2.5% ammonium molybdate in 3 N sulfuric acid was added.
~fo this mixture. 0.2 ml of the reducing agent, 1-amino-2-naphthol sulfonic acid was added.
The collagen mixture was then incubated for 10 minutes at 100°C. and observed for the development of color. as measured at 600 nm in a spectrophotometer ()3eckman DU-2).
Potassium dihydrogen monophosphate was used as a phosphorous standard.
Unmodified collagen prepared according to Example 2 was used as a control.

WO 98130225 PCTlUS98/00371 The phosphorous content was estimated by F:raphing the results obtained with the potassium dihydrogen monophosphate standards. In this experiment, no bound phosphorous was detected in the TTPTM-treated samples. For samples with more phosphorylated collagen (i.r:., where 40°/~ of the modifiable sites available in the collagen were phosphorylated), ?.S%
(wt/wt: dry weight basis) bound phosphorous was detected. For samples with less . phosphorvlation. 0.8% (wt/wt: dry weight basis) bound phosphorous was detected.
These results indicated that there was a significant increase in the phosphorous content uf~ the modified collagen as compared to the untreated control. No measurable amount of phosphorus was detected in the untreated collagen control sample, It was estimated that a considerable number of hydroxy amino acids (e.~,l.. serine and tyrosine) were irreversibly phosphorylated to ibrm phosphoester bonds. Although it is not necessary to understand the invention. Figure 3 shows a reaction that may occur during this phosphorylation.
This modified collagen was shown to possess improved biological characteristics. as described in Examples 10-15. However. it is recognized that care must be taken in the 1 s purification of collagen, as impurities present in impure collagen preparations may engender unfavorable biological reactions due to the phosphorylation of contaminant proteins (e.~~., elastin).
Importantly, the amount of modification can be controlled, ranging from none to a maximum of approximately 40% of the modifiable amino acids present in collagen. The pH
?0 conditions under which the reaction occurs may vary from 8 to 13, depending upon the amount of modification desired. In addition, the reaction temperature may range from 4-4s°C. It was observed in other experiments, that the samples tend to freeze at temperatures less than 4°C. while at temperatures above 45°C. the collagen may become denatured.
The chemical used to react with the collagen may be selected from the group comprisin~~, but not limited to. various polyphosphates (e.~., sodium hexametaphosphate, sodium ultraphosphate. and sodium tetrametaphosphate), phosphoric anhydride, and phosphoryl trichloride solubilized in organic non-polar solvents such as hexane, etc.
It is also contemplated that other non-collagenous biological materials for various biological applications will be chemically modified in a like manner. It is also contemplated s0 that other modifications useful for preparing collagen to be used in cell culture or in virn applications. including, but not limited to tluorination, halogenation (c~.
~., ehlorination), sulfonation. and/or hydroxylation may be performed on collagen prepared according to the methods of Example ?. as well as phosphorylated collagen prepared according to the above described methods. It is contemplated that these modifications be conducted in conjunction (i.c~., multiple modification procedures may be conducted on the same preparation), as well as separately.

Solubility Of Modified Collagen In this Example, the solubility of purified type 1 collagen, modified as described in Example 6 was determined, and compared with the solubility of native collagen..
In this Example, 10 ml of 0.3% solutions of un-modified (i.c~., native) collagen and modified collagen were prepared in 0.1 M acetic acid. The optical density of each solution was determined at 450 nm. A small quantity of 0.5 M NaOH was carefully added to each solution. in order to adjust the pH to neutrality (i.c~., 7). The optical density was again measured at 450 11tH. The following table shows the optical density values for these samples.
1 ~ The increase in optical density of the native collagen indicated that there was more insoluble protein in this sample, as compared to the modified collagen. Thus, the modified collagen was also found to have better solubility features under neutral conditions.
While it is not necessary to understand the present invention, this observation may be the result of a shift in the isoelectric point of the collagen.
en At pH 3.5 And 7 Table .t.
Solubility Of Modified And Unmodified Collag Sample Optical Density At Optical Density At pH 3.5 pH 7 Unmodified Colla~~en 0.001 0.06 Modified Colla~en 0.001 0.01 Preparation Of CollagenPROTM Film In this Example, films (i.e.. membranes) of chemically-treated, filter-sterilized collagen (i.e.. CollagenPROT"'), prepared according to the methods described in Example 6 were s0 prepared for use in in vivo and in vitro experiments. In some experiments, the collaLen was prepared from bovine Achilles tendons, rather than bovine hide. Both collagen sources were found to be satisfactory for use in the following Examples.

First. the suspension was poured into sterile 15 x ? cm petri dishes to a depth of approximately 8 mm. The petri dishes were covered, and stored at a constant temperature of 37°C for ~18 hours, in order to promote uniform initial gelation. This often required 3-~ days for the tllms to visually appear to be dry. At the end of the 48 hour initial gelation phase. the petri dishes were uncovered, and drying was allowed to continue at room temperature in a c:he1111Ca1 ft1i17e hood. At the end of drying, the collagen films were sterilized with ethylene mile ~~~1S and used for animal implantation, transplantation. il'attlllg. or other experiments.
I~hese films were also used in subsequent experiments.

In Vivo Evaluation Of Chemically Modified Collagen In Skin Transplants In this Example. the suitability and biocompatibility for in rive applications of the chemically-modified collagen type I produced as described in Example G.
unmodified collagen prepared as described in Example 2, were determined. Purified. washed collagen samples obtained as described in Example 9, were dried in the form of thin films of 0.3 mm thickness.
in this Example, sixteen adult male Swiss albino mice were used to test the suitability and biocompatibility of these collagen preparations. First. a circular area of 1 cm in diameter ?0 of skin was surgically removed from the backs of the animals. These areas were then covered under aseptic conditions with test or control collagen film ( i. e.. grafted).
The colla~~en used in this Example was sterilized by exposure to ethylene oxide. The animals were observed for gross indications of inflammation (i.e.. redness, swelling, heat. etc.), for a three week period.
No adverse responses were noticed for any of the animals. The animals were then sacrificed at the end of three weeks following the grafting of the collagen film.
The entire skin Clap, including the grafted collagen was removed from each of the animals from the surgical site, and fixed in 70% alcohol, dehydrated, and embedded in wax.
using methods known in the art. Embedded samples were cut into sections S

thickness. and stained with Goldner"s one-step trichrome stain (OST). and hematoxylin and eosin (H & E), using methods known in the art.
The histological results showed no adverse response with either of the two experimental ttroups. Cellular infiltration and the rate of neo-epithelialization (i.e.. epithelial cell infiltration towards the center of the graft) was not affected by any of the test graft materials. In contrast. the control sample showed noticeable numbers of foreign body giant cells attached to the collagen film. In addition, as shown in Figure ?. there was a slower rate of epithelialization observed in the controls.

lir Vivo Evaluation Of Chemically Modified Collagen In Implants In this Example, type I collagen obtained from tissue samples of bovine intestine (obtained ti~om a local market) was purified and chemically modified using the methods of Example G. These samples were then implanted into rabbits, in order to determine the suitability and biocompatibility of collagen prepared according to the methods of the present IIlVentloll. In these experiments. control collagen prepared according to the methods described in Example ? was also used.
1 ~ Adult. male New Zealand white rabbits of approximately 3.5 to 4 kg obtained from the Nitabell Rabittry were used in this Example. The animals were first sedated with an appropriate dosage of l:etamine, and anesthetized with ketamine/xylaxine (PDL) as appropriate for minor surgery. The hair on the dorsal side of the animals was shaved and prepared for surgery with a betadine scrub and a 70% alcohol wipe. The animals were divided into three groups of two animals each. A small incision was made in the skin of each animal, in order to create subcutaneous pockets. Four implantations were made for each group of rabbits. Six of the rabbits (24 sites) were operated on bilaterally, with implants placed on both sides of the dorsal mid-line. Each implant comprised ~0 mg of dried collagen sample were rolled into an approximately round ball and placed subcutaneously at each site.
The animals were observed for three weeks for gross indications of inflammation (e~.,s,~..
redness, swelling, etc.). No adverse responses were observed for any of the animals. After 3 weeks, the animals were sacrificed and the implants were surgically removed.
fixed in 70%
alcohol. dehydrated and embedded in paraffin. using methods commonly known 117 the art.
Sections of ~ micron in thickness were cut. and stained with OST and H & E, using methods commonly known in the art.
The results showed more vascularization and fibroblastic ingrowth in both experimental groups. The control samples had relatively poor vascularization.
as well as a prevalence oi~ multi-nucleated giant cells, reflecting the lesser biocompatibility of these samples.

Alkaline Phosphatase Activity In this Example. the collagen implants tested as described in Example 9 were analyzed For their alkaline phosphatase activity. Alkaline phosphatase activity was of interest. as it has been hypothesized that this enzyme is involved in tissue formation and calcification. The I 0 assay used 111 Ihls Example is based on the methods of Bradenberger and Hanson ( H.
Bradenber~~er and R. l-Ianson, Helv. Chim. Acta 36:900 [1953]; and Hofstee (B.H. Hofstee, Arch. BloChe117. Biophys.. X1:139 [1954]). In this colorimetric method. the effect of alkaline phosphatase on the hydrolysis of o-carboxy-phenyl phosphate is measured.
Briefly. samples of approximately 10 mg from each of the harvested collagen implants 15 from Example 10 were dispersed at a rate of I mg in 1 tnl of Tris buffer (0.1 M Tris, pH 8.5) for five minutes. Care was taken to maintain the temperature of the dispersed samples at 0-~°C. in order to prevent enzymatic reactions from occurring prior to the assay procedure. A
small amount of detergent (to a final concentration of 0.1 M sodium deoxycholate) was added to the dispersed samples, in order to facilitate the release of membrane-bound enzymes. The 20 dispersed samples were then mixed in a Polytron hamogenizer (Brinkmann).
for 30 seconds.
while they were chilled in an ice bath.
For each sample. the solutions were incubated in a water bath at 25°C, for 20-30 minutes in order to allaw activation of the enzymes. A test solution comprised of 2 ml glycine (0.? M, pH 8.8), 1 ml of 3.6~ mM o-carboxy phenyl phosphate (OCCP), and 0.5 ml ?5 of 0.05 M MgCI, was placed into a cuvette, and allowed to incubate for 3-4 minutes at 25°C.
Collagen test samples (0.1 ml, pretiltered using a 0.22 micron filter attached to a ~ ml syringe) were added to the solutions in the cuvettes. The optical density of the cuvette contents was then determined at 300 nm, at room temperature.
The activity is expressed in units per mg of tissue. as based on the number of 30 micromoles of OCCP hydrolyzed per minute at 25°C, at pH. under the conditions described above.

i The results showed siPnificantlv elevated amounts of alkaline: phosphatase (34%
increase: p <0.0005) activity in the modified collagen implants as compared to the uiunodif3ed implants.

Usc Of CullagenPROT"' For Wound Healing In this Example. the suitability of type 1 collagen was extracted from bovine Achilles tendons (obtained from a local market), and made into uniformly thin dry films as described in Example 9. A control film was prepared using the standard pepsin enzyme treatment method described in U.S. Patent No. 5.374,539. In addition to the Collagen PROT'"' described in Example 9. native tvpe I bovine collagen prepared as described in U.S. Patent No. x.374.539, and bovine collagen cross-linked with glutaraldehvde (as described in this patent). were tested. All of these samples were tested without in the presence of 0.~ ml of reconstituted epidermal growth factor (EGF;
Collaborative Research lnc.)( I Etg/3 ml PBS). as well as without the addition of EGF.
Epithelial cell cultures were chosen for this Example, as they provide a model system' for investigating the events that govern the closure of superficial epithelial defects and for assaying the actions of exogenous agents on these events. These methods provide some advantages. as compared to in vivv models of wound closure. For example, it is much easier to manipulate the extracellular milieu in cell culture systems. Prior reports indicate that the in oirro epithelia( response is similar to the response observed ira viva. and can be readily duantitied (.Sec e.,tr.. S. Gunasekaran et al.. Proc. Soc. Biomater., ?0:311 (1994]; and S.
Sirnmons cu crl.. Toxicol. Appl. Pharmacol., 88:13-23 (1987)). This experiment was designed to demonstrate the comparative suitability of various collagen substrata for wound closure. In particular. the interaction of the collagen substrata and EGF were investigated.
Primary cell cultures were established from surgically removed corneas obtained from sacrificed New Zealand white rabbits ( 1.5-2.5 kg). The surgically isolated corneas were incubated in Dispose I1 (Boehrin~er Mannheim) at 37°C, in a humidified CO, incubator.
3U After 1 hour. the corneas were transferred to primary cell culture medium comprised of Dulbecco's modified Eagle's medium enriched with 5% fetal calf serum. 0.5%
dimethyl sulfoxide (DMSO). ~0 n1;/ml lentamicin, 146 ftg/ml L-glutamine. 5 Ly/ml insulin, and l0 ug/ml EGF. The full thickness of the corneal epithelial sheets were gently peeled off the corneal samples and placed in trypsin. gently mixed. and centrifuged at 2.000 xg for ~
minutes at ~l°C. The supernatants were removed and the cells from six corneas were combined and resuspended in fresh primary culture medium and plated in six 35 mm culture dishes. The cultures were placed at 37°C, in a humidified CO, incubator. The culture medium was changed three times per week.
By day 7 of incubation. the primary cultures had grown to contluency and were trypsinized for ~-10 minutes. The cells were pipetted from the culture dishes and centrifuged at 2000 x g for ~ minutes. The supernatant were removed and the control culture medium composed of the Dulbecco's modified Eagle's medium described above, but exclusive of the EGF. The cells were pipetted to yield a suspension of single cells and counted using a hemacytometer. Twenty-four well multiplates were seeded at 3-4 x 10'' cells/well in 1 ml medium per well. The subcultured cells were grown for 7 days, with the medium changed three times durin~~ this period.
On day 7 of subculture, the cells contained in four wells were counted. in order to I ~ ensure that each well had at least I .6 x 10' cells. 'l'he cell layers were injured ("wounded") using filter disks (7 Ixxlxx diameter). as described by Jumblatt and Neufeld (Sve, M. Jumblatt and A. Neui-eld, Invest. Ophthamol. Visual Sci., 15:4-14 [1986]). Briefly, the cell culture medium vas removed from the wells and the filters were placed on top of the cell layer and pressed down with a plunger (7 mm diameter). A pre-chilled steel probe 6 mm in diameter was held for S second on the bottom of the wells, directly below each disk.
Disks with adherent dead cells were then removed from each well. Prior to adding the control medium, the test materials (collagen prepared according to the methods described in Example 9 (CollagenProTM), as well as native type I bovine collagen and cross-linked type I bovine collaben (prepared according to U.S. Patent No. x,374,539). were added to each well. The multiplates containing the "wounded" cell layers and test collagens were re-incubated for 24 hours, to allow the growth of cells into the wounded area. The wells were observed at intervals during this 24 hour growth period. to measure the area of cell layer wounded by the disk. These measurements were taken microscopically, at 0. 12. 18, and 24 hour time intervals.
Closure of the injured area on the cell layer within the wells was stopped by fixation of the cell layers and collagen test samples. with 1(>% neutral buffered formalin. for at least 10 minutes. The cells were then stained with Giemsa stock solution for at least 10 minutes, _37_ rinsed in l0'%~ neutral buffered formalin, and allowed to air drv. The stained wells contained in the microplate were observed microscopically and the wound areas were measured.
Fissure 3 is a bar chart that shows the in vitro wound closure results obtained in this Example. In this Figure, "SC" represents CollagenProT"'. "NC" represents native collagen, "XC" indicates cross-linked type I bovine collagen, and "GF" indicates EGF.
These results indicate that the collagen preparations do not perform in the same manner. The Collu~enfroT~' with and without EGF results showed a faster wound closure rate compared to the results obtained with the other collagen preparations. The ire viva experiments described in Examples 10 and I 1. demonstrated that the delivery of growth factors was more effective 1 U when delivered through Collagenl'roT"', as compared to native type I
collagen prepared according to the method of U.S. Patent No. 5,374,539. In addition, these results indicated that in general. cross-linking of collagen does not assist epithelization.

l5 In Vitro Hemostatic Tests In this Example, collagen prepared as described in Example 6. was used in additional tests to determine its utility in in viva as a hemostatic agent.
Hemostasis (i.e.. blood clotting) is a primary event during the steps involved in normal 20 wound repair. Collagen is a natural hemostatic agent that is present in almost every tissue, and which also appears to have specific affinity for cells and growth factors essential for normal v-ound healing. (,S'ce e.,~~.. Gunasekaran, in Encyclopedia of ~
Biomcrtericrl.s cmc!
l3iocn,~~inecrirr,~l. 2(37):2293-2310 [1995]). Although other materials may assist hemostasis. it is possible that, unlike collagen, they will not optimize the rate of wound healing. 'The object 25 of this Example was to determine the hemostatic and wound healing abilities of various compounds and substances. In this E;cample, collagen fibers (Avitene, obtained from Medchem Products), sponges (Helistat, obtained tcom Collatek), and gelatin (Gelfoam, obtained from Arizona Health Sciences), and gelatin fragments prepared as described below were tested. Additional substances previously used as hemostatic agents. such as thrombin 30 (c~.,~=.. Thrombinar and Thromostat). and cellulose (c~.g.. Surgicel and Oxycel. .f & .1 lvledical) were not included in these experiments. Thrombin was not included as it may have immunological and Local delivery problems.
- 3$ -Native type I bovine collagen was prepared as described in Example ?. Avitene.
Helistat. and Gelfoam were purchased from their respective suppliers. Gelatin fragments were prepared by limited, non-specific protease treatment of gelatin (Sigma). The gelatin fragments were then purified by size exclusion column chromatography on Sephadex G-50 s and G-100 gels in Mufti-Spin columns (Axygen), as described by the manufacturer. Two ~~roups of fi-a~~ments were isolated and purified. One group was designated as "high molecular weight gelatin peptide." This ~~roup contained peptides of molecular weights in the range of s0-100 kD. The other fragment Lroup was designated "low molecular weight gelatin peptide." This group contained peptides with molecular weights in the range of 30-50 kD.
The standard Lee-White clotting method was used in this Example, as described by Brown (B.A. Brown, Hc.~rrra~olngy: Principles and Practice. 3d ed.. Lea &
Febiger, Philadelphia. [ 1980, pgs 125-126). commonly known to those in the hematology art.
~~lultiple experiments were conducted using this method. in which the volume of blood tested was either 0.~ ml or 1 m! (and the other reagents maintained in the proper proportions). As 1 ~ no differences were observed between the tests run with these differing volumes, in most experiments, U.5 m! blood was used. Briefly, 3 mg of each sample previously stored in a desiccator was carefully measured. and each was separately placed into 10 cm long clean polypropylene vials. Fresh blood (5 ml) obtained by venipuncture from healthy humans was collected. and the time of blood collection was noted. To each tube, 0.5 ml of blood was ?0 added and gently agitated every 30 seconds, until clots formed. The time of initial clot formation was noted.
The results of these experiments are shown in Figure 4. As shown in this figure, the clotting time was the greatest for the low molecular weight gelatin peptide fragments ("LMW
Gel. Pept."), and lowest for the CollagenProTM (indicated as "native coll." in this Figure).
25 The high molecular weight gelatin peptide fragments ("HMW Gel. Pept.") time was almost as high as the low molecular weight gelatin peptides. The collagen fibers obtained from Avitene ("Avitene") had the second-most rapid clotting time, while the Helistat ("Helistat") and Gelfoam ("Gelfoam") preparations had the next most rapid times, respectively.
As shown in (: figure ~1. the Gelfoam clotting time was only slightly faster than the high molecular weight ,0 gelatin peptide fragments.

Hemostasis In Vivo This Example, a continuation of the previous example. was conducted in order to assess the hemostatic capabilities of the collagen and other preparations used in Example 9 in ri rn.
In this Example. adult male; Sprague-Dawley rats were used. Circular regions (? cm) uf~ foil-thickness skin were surgically removed from the bacla of anesthetized the animals, as described in Example 10. The exposed areas were aseptically covered with the collagen preparations (i.c'., bratted) described in Example 13. After 3 weeks, the animals were sacrificed and the grafts were surgically removed, fixed in 70% alcohol.
dehydrated and embedded in paraffin. using methods commonly known in the art. Sections of 5 micron in thickness were cut. and stained with OST and H & E. using methods commonly known in the art.
1 ~ The relative extent of neo-epithelization and wound healing were observed and assessed histologically. There were no I;ross signs of inflammation apparent in or around the grafted areas on the animals' backs. The results of this experiment are shown in Figure 4.
As indicated in this figure, the CollagenPROTM film showed a significantly taster wound healing rate and blood clotting time, as compared to the rest of the samples, with Avitene ?0 showing the second fastest rates. and Helistat showing the third fastest rate. The percentage of wound healing and blood clotting time results are shown in Figure 4.
As previously described. these differing substances provide varying results in biological systems. Although it is not necessary to understand the invention.
the relatively poor performance of gelatin and gelatin peptides may indicate that the structure of collagen is ?5 important in the processes of hemostasis and wound healing.
From the above, it is clear that the various embodiments of collagen prepared according, to the methods of the present invention are suitable for various biomedical applications that require non-inflammatory collagen. It is contemplated that the non-30 inflammatory collagen of the present invention be used in multiple settings and for numerous applications. including but not limited to, non-inflammatory collagen sutures.
collagen soft tissue replacements including wound and burn coverings. arterial vessel replacements.
hemostatic absents. dru~~ delivery matrices, vitreous replacement for ophthalmologic therapy.

~i,dodontic therapy, cell culture supports, etc. It is titrther contemplated that various 1:I11bod1I11e17tS OF the present invention will find use in any form.
includin~~. but not limited to fibrous or membrane films, bags, sponges, suture threads. and aqueous suspensions, as well as composite materials. In addition. collagen prepared according to the present invention may be Further modified as necessary for the desired application and to provide an improved bioactive response. It is also contemplated that the methods of the present invention will be applicable to the preparation of other biomolecules as well as collagen.

Claims (26)

CLAIMS:

1. A method for purifying collagen comprising:
a) providing:
i) a sample comprising collagen;
ii) first and second proteolytic enzyme preparations; and ii) a reducing agent;
b) exposing said collagen sample to said first proteolytic enzyme preparation to produce a first collagen solution;
c) exposing said first collagen solution to said reducing agent to produce a second collagen solution;
d) exposing said second collagen solution to said second proteolytic enzyme preparation to produce purified collagen.

2. The method of Claim 1, wherein said first proteolytic enzyme preparation comprises papain.

3. The method of Claim 1 or Claim 2, wherein both of said first and second proteolytic enzyme preparations comprise papain.

4. The method of any one of Claims 1 to 3, wherein said reducing agent is selected from the group consisting of sodium sulfide, dithiothreitol, glutathionine, and sodium borohydride.

5. The method of Claim 4, wherein said reducing agent further comprises an alkali selected from the group consisting of sodium hydroxide, ammonium hydroxide, potassium hydroxide, and calcium hydroxide.

6. The method of any one of Claims 1 to 5, further comprising the step of e) exposing said purified collagen to a delipidation agent to produce delipidated collagen.

7. The method of Claim 6, wherein said delipidation agent comprises a mixture comprising chloroform and methanol.

8. The method of Claim 7, further comprising the steps of f) compressing said delipidated collagen to produce compressed collagen; g) dehydrating said compressed collagen to produce dehydrated collagen; and h) dispersing and drying said dehydrating collagen to form collagen fibers.

9. The method of Claim 7, wherein said delipidated collagen is exposed to a phosphorylating agent to produce phosphorylated collagen.

10. The method of Claim 9, wherein said phosphorylation agent is selected from the group consisting of sodium trimetaphosphate, sodium hexametaphosphate, sodium ultraphosphate, sodium tetrametaphosphate, phosphoric anhydride, and phosphoryl trichloride.

11. The method of Claim 9, wherein said phosphorylation agent comprises sodium trimetaphosphate.

12. The method of any one of Claims 1 to 11, further comprising the step of de-epithelializing said sample prior to exposing said sample to said first proteolytic enzyme preparation.

13. Purified collagen prepared according to the method of Claim 3.

14. A method for production of biocompatible collagen comprising:
a) providing:
i) a sample comprising collagen;
ii) first and second proteolytic enzyme preparations;
iii) a reducing agent;
iv) a delipidation agent; and v) a phosphorylation agent;
b) exposing said collagen sample to said first proteolytic enzyme preparation to produce a first collagen solution;
c) exposing said first collagen solution to said reducing agent to produce a second collagen solution;
d) exposing said second collagen solution to said second proteolytic enzyme preparation to produce a proteolyzed collagen solution;
e) exposing said proteolyzed collagen solution to said delipidation agent to produce delipidated collagen; and f) exposing said delipidated collagen to said phosphorylation agent to produce phosphorylated collagen.

15. The method of Claim 14, wherein said first proteolytic enzyme preparation comprises papain.

16. The method of Claim 14 or Claim 15, wherein both of said first and second proteolytic enzyme preparations comprise papain.

17. The method of any one of Claims 14 to 16, wherein said reducing agent is selected from the group consisting of sodium sulfide, dithiothreitol, glutathionine, and sodium borohydride.

18. The method of any one of Claims 14 to 17, wherein said delipidation agent comprises a mixture of chloroform and methanol.

19. The method of any one of Claims 14 to 18, wherein said phosphorylation agent is selected from the group consisting of sodium trimetaphosphate, sodium hexametaphosphate, sodium ultraphosphate, sodium tetrametaphosphate, phosphoric anhydride, and phosphoryl trichloride.

20. The method of Claim 19, wherein said phosphorylation agent comprises sodium trimetaphosphate.

21. The method of any one of Claims 14 to 20, further comprising the step of de-epithelialization of said sample prior to exposing said sample to said first proteolytic enzyme preparation.

22. The method of any one of Claims 14 to 21, further comprising the step of filter-sterilizing said delipidated collagen prior to exposing said delipidated collagen to said phosphorylation agent to produce phosphorylated collagen.

23. The method of any one of Claims 14 to 22, further comprising the steps of compressing said delipidated collagen to produce compressed collagen; dehydrating said compressed collagen to produce dehydrated collagen; and dispersing and drying said dehydrating collagen to form collagen fibers, prior to phosphorylating said delipidated collagen.

24. Biocompatible collagen prepared according to the method of Claim 16.

25. Use of the purified collagen of Claim 1 for achieving hemostasis.

26. Use of the purified collagen of Claim 1 for transplantation.