Synthesis of cross-links in the helical domain of collagen using pyridoxal 5-phosphate and copper or iron

Aldehyde cross-link intermediates and cross-links are generated in the central helical portion of collagen by incubating collagen with pyridoxal-5-phosphate and either cupric copper ion or ferrous iron ion. The cross-links are chemically similar to natural cross-links found in the non-helical regions and are directly between amino-acid moieties naturally occuring in the central helical portion of collagen. Cross-linking and utilization of aldehyde intermediates occurs when the product is reincubated after pyridoxal is removed. Alternatively maintaining the product at body temperatures will promote cross-linking. The cross-linked collagen product has increased resistance to enzyme degradation.

BACKGROUND OF THE INVENTION 
Collagen is a protein comprising the major fibrous element of mammalian 
skin, bone, tendon, cartilage, blood vessels, and teeth. Its biological 
purpose is to hold cells together in discrete units; and secondarily it 
has a directive role in developing tissues. In mammals, collagen is the 
principal protein and comprises ten percent of the total protein content 
of the body. 
The collagen proteins are distinctive in their physical characteristics in 
that they form insoluble fibers possessing high tensile strength. It is 
the fibrous nature of the collagen that serves to hold the various body 
structures and components together. In fact, the word "collagen" is 
derived from the Greek words meaning "to produce glue". Thus collagen is a 
vitally important biological protein. 
While the molecular structure is modified to meet the needs of particular 
tissues, all collagens are organized into a common structure consisting of 
three polypeptide chains that form a triple stranded helix. These triple 
stranded helical units, in turn, are formed into a quarter-staggered array 
of linearly aligned bundles which make up collagen fibers. The collagen 
fibers are stabilized by covalent cross-links. Two kinds of cross links 
are formed, those which are intramolecular between the helically stranded 
polypeptide chains; and those which are intermolecular between different 
helical units. 
These cross-links principally occur in the non-helical ends of the peptide 
chains wherever lysine, or hydroxylysine amino acid residues occur. Such 
cross-links are generated between the lysine or hydroxylysine residues 
through either an aldol condensation or Schiff base reaction. In the first 
type of reaction the .epsilon.-amino group of a lysyl residue is converted 
into aldehyde by the enzyme, lysyl oxidase. If sufficiently adjacent one 
another, two such aldehydes undergo an aldol condensation to form an aldol 
condensation product. In the second type of reaction, aldehydes derived 
from lysyl or hydroxylysyl groups can also condense with the 
.epsilon.-aminogroup of lysyl or hydroylysyl residues to form Shiff base 
cross-links. 
In some instances the aldol-lysine cross-links can react with a histidine 
side chain to form an histidine-aldol cross-link. In other instances, the 
aldehyde group in the histidine-aldol cross-link can, in turn, form a 
Schiff base with yet another side chain, e.g., hydroxylysine. By such 
cross-linking reactions, four side chains and two or more molecules can be 
covalently bonded together. 
It has been shown that purified collagen can be utilized medically in 
reconstructive and cosmetic surgery for the replacement of bony structures 
or gaps in bony structures, and for filling out tissues where wrinkles 
have formed. In such usage, collagen is secured from mammalian sources, 
e.g., calves, and extraneous proteinaceous material is removed by various 
dissolution, precipitation and filtration techniques to leave a pure 
collagenous product. Native collagen has limited clinical usefulness since 
it may induce antigenic response in the host subject. Such response is 
generated principally by the non-helical terminal portions of the collagen 
molecule. These end regions can be cleaved by treatment with a proteolytic 
enzyme, e.g. pepsin. After digestion with pepsin, the cleaved peptide ends 
are discarded and only the helical domain which comprises more than 95% of 
the molecule remains. These molecules are of low antigenicity and they can 
be used for the purposes noted above without undue antigenic side effects. 
Unfortunately, however, the helical collagen domain contains little, if 
any, of the native covalent cross-links that stabilize native collagen and 
produce its high tensile strength as well as resistance to degradation by 
enzymes and resorption by body fluids. Thus, the unmodified collagen 
products available to the medical profession are soon subject to 
break-down and resorption in a host subject unless artificial, potentially 
antigenic cross-links are introduced. 
It is therefore desirable to devise a low-antigenic collagen product that 
will exhibit the tensile properties of native collagen and resist 
degradation and resorption. One obvious technique for achieving such a 
desired collagen would be the induction of cross-links between various 
amino-acid residues occuring in the central portions of the collagen 
helixes. The nature of such cross-links have been noted above. However, 
such cross-links normally occur in those portions of the collagen that are 
cleaved off to yield a low-antigenic product. 
Some reports have noted the induction of cross-links in the low-antigenic 
helical collagen by employing glutaraldehyde as an intermediate moiety in 
the production of cross-links between lysine residues. However, the 
introduction of glutaraldehyde into the collagen cross-links introduces a 
new antigenic determinant, and may alter certain physical properties of 
cross-linked fibrils in undesirable ways. 
It is of considerable interest, therefore, to increase the resistance of 
low-antigenic collagen to degradation when emplaced within body tissues. 
Such improvement could be provided by inducing native-type cross-linking 
into the helical low antigenic domain of the collagen molecule. 
BRIEF DESCRIPTION OF THE INVENTION 
The present invention relates to the production of native cross-link 
intermediates and cross-links in the helical domain of collagen. The 
material in which native cross-links are induced is collagen obtained from 
any suitable animal source, which is purified to remove non-collagenous 
materials; and is thereafter treated, as by proteolytic enzymes, e.g., 
pepsin, to remove the antigenic ends from the collagen molecules. Such 
collagen, while retaining the helical structure and fiber forming 
properties of native collagen, lacks the native cross-links that provide 
native collagen with its high tensile strength and great resistance to 
degradation by enzyme and resorption by body fluids. 
As used herein "native" or "native-type" cross-links, or cross-link sites 
refers to cross-links directly between amino-acid moieties that normally 
occur in the collagen peptide chains. Such native cross-links principally 
occur between lysine or hydroxylysine residues. The cross-linking "sites" 
are those lysine or hydroxylysine residues that have been altered into the 
aldehyde form and are thereby available to produce cross-links by Schiff 
base formation. 
More specifically, the present invention comprises a method for generating 
native cross-links in the helical region of collagen after the non-helical 
ends have been cleaved by pepsin. Collagen is incubated with 
pyridoxal-5-phosphate and ionic copper or iron to produce collagen 
containing aldehyde cross-link intermediates and cross-linked collagen. 
In the invention process, pure collagen that has had the non-helical 
antigenic ends removed by pepsin digestion, is incubated with pyridoxal 
5-phosphate in the presence of either cupric or ferrous ions. Incubation 
is carried out at a temperature between 25.degree. C. and 37.degree. C. in 
a solution buffered to about a pH of 6.4 to 6.7%. 
Incubation is continued until the lysine or hydroxylysine residues are at 
least partially converted into the aldehyde cross-link intermediate form 
in which the aldehyde group probably forms a Schiff base with pyridoxal. 
Pyridoxal is then removed by dialysis and cross-links can be formed upon 
further incubation of the collagen fibrils, or when emplaced into a 
subject at body temperatures. Such subsequent cross-linking occurs by a 
Schiff base condensation reaction. 
It is therefore an object of the invention to produce native type 
cross-links in the helical portion of collagen. 
It is another object of the invention to induce native type cross-links or 
cross-link sites in the helical portion of collagen by treating the 
collagen with pyridoxal 5-phosphate in the presence of cupric ion or 
ferrous ion. 
It is another object of the invention to produce low-antigenic helical 
collagen having intermolecular cross-links directly between some of the 
amino acid moieties thereof. 
Other objects and advantages of the invention will become apparent from a 
review of the drawing, subsequent specification and the claims appended 
hereto.

DETAILED DESCRIPTION OF THE INVENTION 
Cross-links, or aldehyde cross-linking sites are produced on collagen by 
incubating normally uncross-linked enzyme digested collagen with pyridoxal 
phosphate in the presence of cupric or ferrous ions. Cross-linking 
production is more efficient in the presence of cupric ion, and therefore 
its use is preferred. Nonetheless, ferrous ion is also useful, though 
somewhat less (about 50%-60%) efficient. 
Cross-linking is produced, or aldehyde cross-linking sites are produced, by 
incubating the collagen with the pyridoxal phosphate and cupric or ferrous 
ion at a temperature between about 25.degree. and 37.degree. C. The 
reaction mixture is maintained at a roughly neutral pH, i.e., about 6-7; 
ideally 6.4. Cross-link production occurs more rapidly at the upper end of 
the temperature limit; however, at 37.degree. C. collagen is approaching 
its denaturation temperature. Therefore, it is often advantageous to carry 
out the incubation at somewhat lower temperatures to avoid any heat 
degradation of the collagen. 
During the course of incubation some of the lysine, or hydroxylysine 
moieties in the collagen helix have their side chains converted to the 
corresponding aldehyde, i.e., 
##STR1## 
These lysyl or hydroxylysyl aldehyde residues are then capable of 
cross-linking with other converted lysyl or hydroxylysyl residues by a 
Schiff base reaction, i.e., 
##STR2## 
thus cross-linking adjacent collagen chains. The initial reaction product 
is probably a Schiff base with pyridoxal. The aldehydes are generated and 
pyridoxal removed by dialysis and the Schiff base cross-links are formed 
by reincubation. 
It is to be noted that the by-product of the cross-linking reaction is 
water. Therefore if the lysine is previously tagged at the C-6 carbon atom 
by means of a suitable radio-isotope, i.e., tritium (.sup.3 H or T), the 
water resulting from the cross-linking reaction can be detected in the 
reaction liquid medium. This permits use of radio-assays to assess the 
aldehyde generation that takes place in the invention process. 
As noted above, the preferred collagen for use in the invention consists of 
helical collagen bundles from which the non-helical end portions have been 
removed. The removal of the non-helical ends can be effected in a standard 
procedure wherein a proteolytic enzyme, e.g., pepsin, is allowed to react 
with previously purified collagen, most usually obtained from a bovine 
source. The pepsin cleaves the non-helical ends from the collagen while 
the helical intermediate (and non-cross-linked) portion remains 
unaffected. The desired helical collagen is then separated from the 
digested ends and the enzyme to yield pure helical collagen. Such a 
product is available from commercial sources, e.g., the Collagen 
Corporation of Palo Alto, Calif. 
In the invention process, the low-antigenic collagen is reacted with 
pyridoxal-5-phosphate in the presence of either cupric or ferrous ions. 
The reaction is carried out by incubating the collagen, 
pyridoxal-5-phosphate and metal ion at temperatures in the 25.degree. to 
37.degree. C. range in an aqueous medium maintained at a pH of about 6-7. 
Buffering materials, such as sodium acetate, are added to the medium to 
maintain the desired pH during the incubation. 
The presence of both pyridoxal-5-phosphate and the metal ion are vital to 
the production of the aldehyde intermediates and/or the cross-linked 
collagen. 
Pyridoxal-5-phosphate has the structure: 
##STR3## 
It constitutes the prosthetic group of all transaminase enzymes in 
biological systems. In such systems pyridoxal-5-phosphate faciliates the 
reversible transfer of an amino group from an .alpha.-amino acid to an 
.alpha.-keto acid as its primary catalytic function. In some instances 
enzyme bound pyridoxal-5-phosphate enters into catalytically driven 
decarboxylations, deaminations, racemizations, and aldol cleavages. In 
other instances, pyroxidol-5-phosphate enzymes catalyse reactions at the 
.beta. or .gamma. carbon atom of .alpha.-amino acids both by way of 
elimination or replacement of functional groups. 
In any event, pyridoxal-5-phosphate is a necessary component in the 
cross-linking incubation. Pyridoxal-5-phosphate can be obtained from 
commercial sources, e.g., Calbiochem. 
Although pyridoxal-5-phosphate is a known promoter for transaminations, 
decarboxylations, deaminations, etc., as noted above, it does not catalyze 
aldehyde formation when it alone is incubated with collagen. To achieve 
reasonable development of aldehyde cross-linking sites, it is necessary to 
include either copper or iron ions along with the pyroxidal during 
collagen incubation. 
The mechanism by which the pyroxidal-5-phosphate and cupric or ferrous ion 
act to produce the cross-linking sites is not known, but suffice to say, 
an appreciable number of such sites are produced when low-antigenic 
collagen is incubated with pyroxidal-5-phosphate and cupric or ferrous ion 
under the conditions as herein set forth. 
The cupric or ferrous ion may be supplied from any water soluble source of 
the metal. Soluble inorganic compounds, i.e., metal salts are particularly 
suitable for this purpose. Thus cupric sulfate or ferrous sulfate may be 
used to supply the metal ions. Pure salts of these metals are obtainable 
from any number of commercial sources. 
The collagen is treated by incubation along with the pyridoxal-5-phosphate 
and the ionic copper or iron. The collagen is suspended in an aqueous 
solution and pyridoxal-5-phosphate and ionic copper or iron is added 
thereto. The aldehyde or cross-linking reaction most advantageously occurs 
when the pyridoxal-5-phosphate concentration is maintained at about 0.001 
M. Ionic copper or iron concentrations are optimally maintained at about 
0.002 M. The concentrations of the pyridoxal and metal ions may be varied 
from those noted above; however studies have shown that the production of 
cross-linking sites is most efficient at, or about the noted 
concentrations. 
FIG. 1 of the drawing illustrates that the greatest production of aldehyde 
cross-link intermediates (as measured by tritium release) occurs when the 
cupric or ferrous ion concentration reaches about 0.002 M. At 
concentrations above or below the optimum, the rate of aldehyde production 
is greatly reduced. It is to be understood that lower or higher metal ion 
concentrations will result in aldehyde formation; however such production 
is less efficient. Increased incubations times would be necessary to 
produce the same extent of aldehyde formation as would be produced in much 
shorter incubation times with optimum metal ion concentrations. 
FIG. 2 of the drawing illustrates that optimum aldehyde site production 
occurs when the pyridoxal-5-phosphate concentration is about 0.001 M. At 
lower concentrations, aldehyde production falls off very rapidly. At 
higher concentrations aldehyde production is also reduced; but the drop is 
not as drastic as in the case of lower concentrations. For efficiency, 
i.e., shortest incubation times, the 0.001 M concentration of 
pyridoxal-5-phosphate is preferred. 
The pH at which incubation takes place also affects the rate of aldehyde 
cross-link intermediate formation. In a series of experiments, pepsinized 
chick calvaria collagen was incubated at 37.degree. C. for 2.5 hours with 
CuSO.sub.4 (0.002 M) and pyridoxal-5-phosphate (0.001 M) in 0.1 M sodium 
acetate. The pH of the incubation mixture was changed in each successive 
incubation sample from a low of about 5.5 up to a high of 11.5 by the 
addition of HCl or NaOH as necessary. FIG. 3 of the drawing summarizes the 
results. As will be noted in the Figure, maximum aldehyde production took 
place at a pH of about 6.4. At pH's much below or above 6.4, formation was 
reduced. Therefore, it is desirable to conduct the incubation at a pH of 
about 6.4 or as close thereto as possible. 
Incubation temperatures also influence the rate of cross-linking and/or 
aldehyde production. Generally speaking cross-linking and/or aldehyde 
production remains quite slow at temperatures below about 25.degree. C. 
Above 25.degree. C., the production rate increases quite rapidly; and 
continues to increase at least to 37.degree. C. Since collagen begins to 
denature above 37.degree. C., it is not desirable to use higher 
temperatures. 
FIG. 4 of the drawing illustrates the effect of incubation temperature upon 
aldehyde formation. In FIG. 4, samples of T-6 lysine radiolabelled 
pepsinized chick calvaria collagen were incubated for 3 hours in the 
presence of 0.002 M CuSO.sub.4, and 0.001 M pyridoxal-5-phosphate at 
various temperatures. The results, as measured by tritium release from the 
T-6 lysine are shown in the plot. The increasing rate of aldehyde 
production can be readily noted as incubation temperatures increased to 
37.degree. C. from 25.degree. C. 
The components must be incubated (under the conditions noted above) for 
considerable periods of time in order to create a suitable population of 
aldehyde intermediates and cross-links in collagen. Incubation times of 8 
hours and up to perhaps 24 hours, or more, are required to produce an 
average of two cross-links per molecule. Although the number of 
cross-links produced per molecule may seem small, this low number is 
sufficient to significantly increase the collagen's resistance to 
degradation by collagenase. The treated collagen also becomes less soluble 
in acetic acid; which is a further measure of resistance to resorption in 
a tissue environment. 
To generate cross-links it is often useful to conduct a first incubation, 
and then follow with dialysis to remove pyridoxal and minerals and then do 
a second incubation. It is believed that a relatively brief, e.g. 8 hours, 
first incubation period serves to produce aldehydes as noted above. This 
collagen will then cross-link upon additional incubation after dialysis in 
the absence of the metal ion and pyridoxal-5-phosphate. Studies have shown 
that such double incubated collagen tends to have more resistance to 
collagenase and less solubility after a first relatively short incubation 
time with the metal ion and pyridoxal, and a second longer, e.g., 24 hours 
incubation period after the metal ion and pyridoxal have been removed. 
To illustrate the effect of a first incubation followed by a second 
incubation, in several studies pepsinized radio-labelled chick calvaria 
collagen was first incubated at 37.degree. C. with 0.002M ferrous sulfate 
and 0.001 M pyridoxal-5-phosphate for various periods of time from 4 to 48 
hours. The samples were then dialyzed against 0.5 M acetic acid and 
phosphate buffered saline to remove the ferrous sulfate and pyridoxal and 
redialyzed to initial incubation conditions. The collagen samples were 
then subsequently incubated at 37.degree. C. with no additions to the 
incubation solution. The second incubations were carried out for either 24 
hours or 48 hours. As a control a second set of samples were incubated two 
times for the same periods and at the same temperatures. However, the 
incubations were carried out in the absence of both FeSO.sub.4 and 
pyridoxal. 
To test for the collagen's resistance to degradation, one group of samples 
was lyophilized and then digested by bacterial collagenase at 37.degree. 
C. for 2 hours. The amount of collagen found in the supernatant liquid was 
a measure of the collagen's resistance to enzyme digestion. FIG. 5 of the 
drawing indicates the results. As will be noted therefrom, the ferrous ion 
and pyridoxal treated collagen demonstrated improved resistance to enzyme 
digestion where the first digestion with the ferrous ion and pyridoxal 
continued for up to 24 hours. 
In a second test pepsinized chick calvaria collagen was incubated under the 
same conditions and for the same time periods as in the test noted 
immediately above. However, in these tests, the incubated product was 
lyophilized and then extracted with 0.5 M acetic acid at 23.degree. C. for 
2 hours. The amount of collagen appearing in the extraction liquid was a 
measure of the treated collagen's solubility. FIG. 6 of the drawing 
illustrates the results. It shows that collagen incubated for up to 24 
hours with ferrous ion and pyridoxal demonstrates considerably less 
solubility than does collagen incubated in the absence of metal ion and 
pyridoxal. 
In all instances, the metal ion and pyridoxal are removed from the collagen 
after the incubation is terminated. Removal can be effected by dialyzing 
the reaction mixture. Thus the metal ion and pyridoxal can be separated 
from the collagen by dialyzing against a suitable salt or acid solution, 
e.g., 0.5 M acetic acid. Upon dialysis the metal ions and 
pyridoxal-5-phosphate diffuse across the membrane barrier while the 
treated collagen remains behind. Aldehydes that are bound to pyridoxal are 
regenerated and able to cross-link. The material can then be reincubated 
to promote cross-link formation. The suspension of collagen with its 
induced cross-links and/or aldehyde intermediates (cross-linking sites) is 
then available for use. 
It will be understood that cross-linking occurs either during the second 
incubation after dialysis or when the collagen is emplaced within the 
tissues of a patient. Until such use, the treated collagen may be 
refrigerated to prevent further cross-linking.