Aqueous atelocollagen solution and method of preparing same

An aqueous atelocollagen solution, which can be injected into living bodies as a medical material, has a pH value in the range from about 6.5 to about 8.0 and an osmolality in the range from about 250 to about 320 mOsm/KgH.sub.2 O, and contains a phosphate buffer solution, or glucose and a phosphate buffer solution, as an agent to adjust pH and osmolality within the ranges specified above. This aqueous atelocollagen solution can be prepared by dissolving atelocollagen in an aqueous acidic solution and adding the above-mentioned pH- and osmolality-adjusting agent to the resulting solution in such an amount as to adjust pH and osmolality of the final solution within the ranges mentioned above.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to an aqueous solution of atelocollagen and the 
method of preparing the same. 
The aqueous atelocollagen solution of this invention can be injected into 
living bodies without any trouble; it is sufficiently fluid to allow easy 
injection, for example, through a fine syringe needle and is capable of 
regenerating collagen fiber when it becomes equilibrated with biological 
conditions. 
2. Description of the Prior Art 
Collagen is the major protein which constitutes the connective tissues of 
animals, such as the skin, blood vessels, cornea, tendons, bones and 
teeth, having a molecular weight of approximately 300,000. It has a 
rod-like molecular structure of helical configuration consisting of three 
polypeptide chains, about 3,000 .ANG. in length and about 15 .ANG. in 
diameter. 
Collagen molecules, which are biochemically synthesized by 
collagen-producing cells in living bodies, are incorporated in the 
surrounding collageous fiber tissue and gradually change into insoluble 
collagen through intermolecular crosslinking. 
This insoluble collagen cannot be extracted by dilute aqueous acid 
solutions. The juvenile collagen immediately after formation, on the other 
hand, can be extracted by dilute aqueous acid solutions because of the 
absence of crosslinks, even when it is present on or in the surrounding 
collagen fiber tissue. This acid-extractable collagen is called soluble 
collagen. 
The insoluble collagen, if treated with a proteolytic enzyme, such as 
pepsin, undergoes fission at the intermolecular crosslinks and becomes 
soluble in dilute acids. During this treatment with pepsin, the 
telopeptide groups at both terminals of each collagen molecule are 
digested, thus leaving collagen with no telopeptide terminal ends. The 
collagen thus released is called atelocollagen. Since the telopeptide 
moiety is primarily responsible for antigenicity of collagen, 
atelocollagen has little antigenicity if any, which makes it very suitable 
for use as a medical material. 
Use of purified collagen as a medical material for therapeutical 
applications has been expanding and becoming diversified. For example, 
purified collagen is employed as local hemostatics in the form of powder 
or sponge, as a traumatic cover and artificial eardrum in the form of a 
membrane or nonwoven fabric, and as contact lenses in a shaped form. It is 
also used in a molded form containing a medicine for slow release 
preparations. In these applications collagen is used in solid form. 
However, there are other fields in which use of collagen in a fluid 
solution state is desired. For example, there has been a demand for an 
aqueous solution of collagen that can be injected into ruptured tissues, 
for example, through a syringe needle to fill up the affected area without 
incision. In this case it is necessary that the collagen solution remain 
fluid during injection without forming fibrous structure, and that the 
collagen molecules contained in it become oriented into fiber form when 
exposed to, and equilibrated with, biological conditions after injection 
to produce bundles of collagen fiber. If such an aqueous solution of 
collagen be developed, the bundles of collagen fiber formed in living 
bodies will accept entry of cells and minute blood vessels from the 
surrounding tissue, and will maintain its volume for a sufficient period 
of time, thus aiding in filling up the injured part. 
Use of soluble collagen or atelecollagen is indispensable to the 
preparation of a collagen solution having such properties. Collagen of 
these types is soluble in a dilute acid solution with a pH value of 5 or 
lower, but such an acidic solution cannot be applied to living bodies. For 
a collagen solution to be applicable to living bodies, preferably it has 
properties close to biological conditions, namely, a pH value of about 7 
and an osmolality of about 280.about.290 mOsm/KgH.sub.2 O, and is 
preferably capable of being injected, for example, through a fine syringe 
needle. The acidic collagen solution mentioned above, however, tends to 
produce collagen fiber before injection under the conditions close to 
those in living bodies described above, and cannot be applied, for 
example, through a fine syringe needle. 
OBJECTS AND SUMMARY OF THE INVENTION 
Thus an object of this invention is to provide an aqueous solution of 
atelocollagen useful as a medical material, which remains fluid under the 
pH and osmolality conditions close to those in living bodies as described 
above and which forms collagen fiber when it becomes equilibrated with 
biological conditions, for example, when it is injected into living 
bodies. 
Another object of this invention is to provide a method of preparing such 
an aqueous solution of atelocollagen which comprises adding a pH- and 
osmolality-adjusting agent to a solution of atelocollagen in an aqueous 
acid, said adjusting agent being preferably a phosphate buffer or a 
combination of a phosphate buffer with glucose.

DETAILED DESCRIPTION OF THE INVENTION 
The aqueous solution of atelocollagen of this invention has a pH value 
ranging from about 6.5 to about 8.0 and an osmolality in the range from 
about 250 to about 320 mOsm/KgH.sub.2 O. 
A phosphate buffer solution selected from the group consisting of 
combinations, KH.sub.2 PO.sub.4 --K.sub.2 HPO.sub.4, KH.sub.2 PO.sub.4 
--Na.sub.2 HPO.sub.4 and NaH.sub.2 PO.sub.4 --Na.sub.2 HPO.sub.4, may be 
used as an agent to adjust the pH of the aqueous atelocollagen solution of 
this invention within the range between about 6.5 and about 8.0. The 
preferable concentration of the phophate buffer in the atelocollagen 
solution is in the range from 0.09 to 0.12M. If the buffer concentration 
is adjusted within this range, the osmolality of the atelocollagen 
solution will be nearly equal to that in living bodies. In addition, other 
solutes may be added to the atelocollagen solution to adjust osmolality 
within the range specified above, and glucose was found to be the most 
preferable solute for this purpose. 
It was proved that addition of glucose causes no regeneration of collagen 
fiber in the atelocollagen solution. 
For example, the osmolality of an aqueous atelocollagen solution with its 
pH controlled at 7.3 by a phosphate buffer of 0.05M concentration was 140 
mOsm/KgH.sub.2 O. No regeneration of collagen fiber was observed in this 
solution when its osmolality was increased to 306 mOsm/KgH.sub.2 O by 
addition of glucose to 3% concentration, or increased to 251 
mOsm/KgH.sub.2 O by addition of glulcose to 2% concentration. In all of 
these cases, the atelocollagen solution could be injected into living 
bodies through a fine syringe needle without any trouble, and became 
equilibrated with biological conditions after injection, regenerating 
collagen fiber. 
The aqueous solution of atelocollagen of this invention can be prepared by 
dissolving atelocollagen in an aqueous acid solution with a pH value of 
preferably 4.5 or lower, most preferably, in the range from 3.0 to 4.0, 
and adjusting the pH of the resulting solution in the range from about 6.5 
to about 8.0 and its osmolality in the range from about 250 to about 320 
mOsm/KgH.sub.2 O, by addition of a pH- and osmolality-adjusting agent. 
The concentration of atelocollagen in said aqueous acid solution may be in 
the range from 0.2 to 10%, preferably from 1 to 8%. 
As examples of said acid solution may be mentioned aqueous solutions of 
inorganic and organic acids, such as hydrochloric, acetic, citric and 
lactic acids, but an aqueous solution of hydrochloric or acetic acid is 
most preferable. 
As the pH- and osmolality-adjusting agent may be used a phosphate buffer 
solution selected from the group consisting of combinations, KH.sub.2 
PO.sub.4 --K.sub.2 HPO.sub.4, KH.sub.2 PO.sub.4 --Na.sub.2 HPO.sub.4 and 
NaH.sub.2 PO.sub.4 --Na.sub.2 HPO.sub.4. The concentration of these 
phosphate buffer solutions before addition may be 0.10M or higher, 
preferably in the range from 0.1 to 0.6M. The preferable mixing ratio of 
said phosphate buffer solution and said acidic solution of atelocollagen 
is such that the concentration of the former after mixing will be in the 
range from 0.09 to 0.12M. It is important that the osmolality of the 
resulting solution be as close to that in living bodies as possible. If an 
above-mentioned phosphate buffer solution of 0.10M concentration is used, 
the osmolality of the resulting solution will be 280 mOsm/KgH.sub.2 O, a 
level very similar to that in living bodies. 
In addition to the above-specified phosphate buffer solution, the aqueous 
atelocollagen solution of this invention may include other solutes as 
osmolality adjusting agent. The most preferable solute used for this 
purpose is glucose. This solute must be added in such an amount as to give 
an osmolality in the range from 250 to 320 mOsm/KgH.sub.2 O to the final 
solution. For example, it was demonstrated that glucose must be added to 
3% concentration when a 0.05M phosphate buffer solution is used, and to 4% 
concentration when a 0.025M buffer solution is employed. When glucose is 
used in combination with a phosphate buffer solution, it is preferable to 
first add glucose to the acidic atelocollagen solution, mix the solution 
well, and then add the phosphate buffer solution, followed by thorough 
mixing. 
The water, phosphate buffer solution and glucose used for the preparation 
of the aqueous atelocollagen solution of this invention are all 
pyrogen-free. To this end purified water obtained by reverse osmosis or 
double-distillation process commonly used may be employed. 
The following examples will further illustrate the present invention. 
EXAMPLE 1 
A portion of the skin was peeled off from the back of a calf, its periphery 
was cut out, the remainder was washed with tap water to remove dirt and 
dust from the surface, and finally washed with pyrogen-free water. The 
calf hide thus obtained was immersed in 70% ethanol, and all hair and the 
uppermost surface of the skin were then sliced off with a razor blade, 
taking care not to leave any hair roots and to keep the exposed surface 
clean. The back surface was also treated in the same way, thus isolating a 
clean piece of calf derma. This was immersed in 70% ethanol overnight, 
excess ethanol was removed and the derma was pulverized under germ-free 
condition. It was then washed with pyrogen-free, 5% aqueous NaCl solution, 
dehydrated by centrifugation, washed with pyrogen-free water, and immersed 
in 70% ethanol overnight. The excess alcohol was centrifugally removed, 
the resultant powder of derm was put in a germ-free dissolver, and a 
solution of pepsin in pyrogen-free water, freed from germs by filtration, 
was added. The amount of pepsin added was 0.5% relative to the powdered 
derma on dry basis. The concentration of powdered derm was adjusted to 
about 0.5%, pH was cotrolled at 3 with hydrochloric acid, and the mixture 
was held at 20.degree. C. for three days with mild stirring to completely 
dissolve the insoluble collagen of the derma. The atelocollagen solution 
thus obtained was successively passed through three filters having pore 
size of 1 .mu.m, 0.65 .mu.m and 0.45 .mu.m, respectively, and the pH of 
the filtrate was raised to 11.0 by addition of caustic soda solution to 
deactivate the pepsin. The pH of the resulting solution was then adjusted 
to 7.0 by addition of hydrochloric acid, the precipitated atelocollagen 
was collected with a centrifugal separator and washed with pyrogen-free 
water, the precipitate was again dissolved in pyrogen-free hydrochloric 
acid, and the pH of the resulting solution was adjusted to 7 to 
precipitate atelocollagen. The germ- and pyrogen-free product thus 
obtained was dissolved in a pyrogen-free hydrochloric acid of pH 3.0 to a 
concentration of 3%. 
To 100 parts by weight of this atelocollagen solution was added 50 parts by 
weight of 0.3M KH.sub.2 PO.sub.4 --Na.sub.2 HPO.sub.4 buffer solution (pH 
7.4). The atelocollagen solution thus prepared was germ- and pyrogen-free 
and contained 2% atelocollagen, with its buffer solution concentration, pH 
and osmolality being 0.1M, 7.3 and 280 mOsm/KgH.sub.2 O, respectively. 
This solution caused no precipitation of collagen fiber when allowed to 
stand at room temperature, and could be easily passed through a fine 
syringe needle. When mixed with a 0.9% NaCl solution (physiological saline 
solution ) or equilibrated with biological conditions, it became turbid at 
once as a result of collagen fiber formation. This is indicative of the 
suitability of this solution for injection application. 
EXAMPLE 2 
A 5% atelocollagen solution of pH 3 was prepared using germ- and 
pyrogen-free atelocollagen obtained as precipitate in the same way as in 
Example 1. To 100 parts (by weight) of this solution was added 50 parts by 
weight of 12%, pyrogen-free aqueous solution of glucose, followed by 
addition of 50 parts by weight of 0.2M, pyrogen-free NaH.sub.2 PO.sub.4 
--Na.sub.2 HPO.sub.4 buffer solution (PH 7.4). This operation was 
performed under germ-free condition. The 2.5% atelocollagen solution thus 
prepared contained 0.05M NaH.sub.2 PO.sub.4 --Na.sub.2 HPO.sub.4 and 3% 
glucose, with its pH and osmolality being 7.25 and 306 mOsm/KgH.sub.2 O, 
respectively. 
This solution caused no precipitation of collagen fiber and could be easily 
passed through a fine syringe needle. When mixed with a 0.9% physiological 
saline solution or exposed to biological conditions, it became turbid at 
once as a result of collagen fiber formation. This is indicative of the 
suitability of this solution for injection application. 
EXAMPLE 3 
A 6% atelocollagen solution with its pH controlled to 3 with hydrochloric 
acid was prepared using the germ- and pyrogen-free atelocollagen obtained 
in Example 1. Under germ-free condition, 50 parts (by weight) of 4% 
aqueous glucose solution was added to 100 parts by weight of the 
atelocollagen solution obtained above, followed by addition of 50 parts by 
weight of 0.32M KH.sub.2 PO.sub.4 --K.sub.2 HPO.sub.4 buffer solution. The 
resultant solution contained 1% glucose and 0.080M buffer solution, and 
showed a pH value of 7.30. This neutral atelocollagen solution caused no 
precipitation of collagen fiber at room temperature, and could be easily 
passed through a fine syringe needle. It regenerated collagen fiber when 
mixed with a physiological saline solution or exposed to biological 
conditions, indicating that it is suitable for injection application.